Pap mutants that exhibit anti-viral and/or anti-fungal activity in plants

ABSTRACT

Disclosed are PAP mutants having reduced phytotoxicity compared to wild-type PAP, and which confer broad spectrum resistance to viruses and/or fungi in plants. One group of PAP mutants is characterized by at least one amino acid substitution in the N-terminus of mature PAP, such as the Glycine 75 residue or the Glutamic acid 97 residue; two groups of additional PAP mutants are characterized by truncations in the N-terminal region of mature PAP and truncations or amino acid substitutions in the C-terminal region of mature PAP, respectively; and a further group are enzymatically inactive which still exhibit anti-fungal properties. Also disclosed are DNA molecules encoding the PAP mutants, mutant PAP DNA constructs, and transgenic seed and plants containing the DNAs. Further disclosed are methods for identifying PAP mutants having reduced phytotoxicity, as well as isolated and purified PAP mutants identified by the method.

This Application is a Continuation of PCT/US96/11546 filed Jul. 11,1996, which is a Continuation-In-Part of U.S. application Ser. No.08/500,611 filed Jul. 11, 1995, now U.S. Pat. No. 5,756,322, andapplication Ser. No. 08/500,694 filed Jul. 11, 1995, now U.S. Pat. No.5,880,329.

TECHNICAL FIELD

This invention relates generally to agricultural biotechnology, and morespecifically to methods and genetic materials for conferring resistanceto fungi and/or viruses in plants.

BACKGROUND OF THE INVENTION

The subject of plant protection against pathogens remains the area ofutmost importance in agriculture. Many commercially valuableagricultural crops are prone to infection by plant viruses and fungicapable of inflicting significant damage to a crop in a given season,and drastically reducing its economic value. The reduction in economicvalue to the farmer in turn results in a higher cost of goods toultimate purchasers. Several published studies have been directed to theexpression of plant virus capsid proteins in a plant in an effort toconfer resistance to viruses. See, e.g., Abel et al., Science 232:738-43(1986); Cuozzo et al., Bio/Technology 6:549-57 (1988); Hemenway et al.,EMBO J. 7:1273-80 (1988); Stark et al., Bio/Technology 7:1257-62 (1989);and Lawson et al., Bio/Technology 8:127-34 (1990). However, thetransgenic plants exhibited resistance only to the homologous virus andrelated viruses, but not to unrelated viruses. Kawchuk et al., Mol.Plant-Microbe Interactions 3(5):301-07 (1990), disclose the expressionof wild-type potato leafroll virus (PLRV) coat protein gene in potatoplants. Even though the infected plants exhibited resistance to PLRV,all of the transgenic plants that were inoculated with PLRV becameinfected with the virus and thus disadvantageously allowed for thecontinued transmission of the virus such that high levels of resistancecould not be expected. See U.S. Pat. No. 5,304,730.

Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-93 (1993), report theAgrobacterium tumefaciens-mediated transformation of tobacco with a cDNAencoding wild-type pokeweed antiviral protein (PAP) and the resistanceof the transgenic tobacco plants to unrelated viruses. PAP, a Type Iribosome-inhibiting protein (RIP) found in the cell walls of Phytolaccaamericans (pokeweed), is a single polypeptide chain that catalyticallyremoves a specific adenine residue from a highly conserved stem-loopstructure in the 28S rRNA of eukaryotic ribosomes, and interferes withelongation factor-2 binding and blocking cellular protein synthesis.See, e.g., Irvin et al., Pharmac. Ther. 55:279-302 (1992); Endo et al.,Biophys. Res. Comm., 15:1032-36 (1988); and Hartley et al., FEBS Lett.290:65-68 (1991). The observations by Lodge were in sharp contrast toprevious studies, supra, which reported that transgenic plantsexpressing a viral gene were resistant to that virus and closely relatedviruses only. See also Beachy et al., Ann. Rev. Phytopathol. 2:451-74(1990); and Golemboski et al., Proc. Natl. Acad. Sci. USA 87:6311-15(1990). Lodge also reports, however, that the PAP-expressing tobaccoplants (i.e., above 10 ng/mg protein) tended to have a stunted, mottledphenotype, and that other transgenic tobacco plants that accumulated thehighest levels of PAP were sterile. RIPs have proven unpredictable inother respects such as target specificity. Unlike PAP which (asdemonstrated in Lodge, supra), ricin isolated from castor bean seed is1000 times more active on mammalian ribosomes than plant ribosomes. See,e.g., Harley et al., Proc. Natl. Acad. Sci. USA 79:5935-5938 (1982).Barley endosperm RIP also shows very little activity against plantribosomes. See, e.g., Endo et al., Biochem. Biophys. Acta 994:224-226(1988) and Taylor et al., Plant J. 5:827-835 (1984).

Fungal pathogens contribute significantly to the most severe pathogenoutbreaks in plants. Plants have developed a natural defense system,including morphological modifications in their cell walls, and synthesisof various anti-pathogenic compounds. See, e.g., Boller et al., PlantPhysiol 74:442-444 (1984); Bowles, Annu. Rev. Biochem. 52:873-907(1990); Joosten et al., Plant Physiol. 82:945-951 (1989); Legrand etal., Proc. Natl. Acad. Sci. USA 84:6750-6754 (1987); and Roby et al.,Plant Cell 2:999-1007 (1990). Several pathogenesis-related (PR) proteinshave been shown to have anti-fungal properties and are induced followingpathogen infection. These are different forms of hydrolytic enzymes,such as chitinases and β-1,3-glucanases that inhibit fungal growth in vby destroying fungal cell walls. See, e.g., Boller et al., supra;Grenier et al., Plant Physiol. :1277-123 (1993); Leah et al., J. Biol.Chem. 266:1464-1573 (1991); Mauch et al., Plant Physiol. 87:325-333(1988); and Sela-Buurlage Buurlage et al., Plant Physiol. 101:857-863(1993).

Several attempts have been made to enhance the pathogen resistance ofplants via recombinant methodologies using genes encodingpathogenesis-related proteins (such as chitinases and β-1,3-glucanases)with distinct lytic activities against fungal cell walls. See, e.g.,Broglie et al., Science 254:1194-1197 (1991); Vierheilig et al., Mol.Plant-Microbe Interact. 6:261-264 (1993); and Zhu et al., Bio/Technology12:807-812 (1994). Recently, two other classes of genes have been shownto have potential in conferring disease resistance in plants. Wu et al.,Plant Cell 1:1357-1368 (1995), report that transgenic potato expressingthe Aspergillus niger glucose oxidase gene exhibited increasedresistance to Erwinia carotovora and Phytophthora infestans. Thehypothesis is that the glucose oxidase-catalyzed oxidation of glucoseproduces hydrogen peroxide, which when accumulates in plant tissues,leads to the accumulation of active oxygen species, which in turn,triggers production of various anti-pathogen and anti-fungal mechanismssuch as phytoalexins (see Apostol et al., Plant Physiol. 20:109-116(1989) and Degousee, Plant Physiol. 945-952 (1994)),pathogenesis-related proteins (Klessig et al., Plant Mol. Biol.26:1439-1458 (1994)), strengthening of the plant cell wall (Brisson etal., Plant Cell 6:1703-1712 (1994)), induction of systemic acquiredresistance by salicylic acid (Chen et al., Science 162:1883-1886(1993)), and hypersensitive defense response (Levine et al., Cell79:583-593 (1994)).

In addition to the studies on virus resistance in plants, RIPs have beenstudied in conjunction with fungal resistance. For example, Logeman etal., Bio/Technology 10:305-308 (1992), report that a RIP isolated frombarley endosperm provided protection against fungal infection totransgenic tobacco plants. The combination of barley endosperm RIP andbarley class-II chitinase has provided synergistic enhancement ofresistance to Rhizoctonia solani in tobacco, both in v and in vivo. See,e.g., Lea et al., supra; Mauch et al., supra; Zhu et al., supra; andJach et al., The Plant Journal 8:97-109 (1995). PAP, however, has notshown antifungal activity in vitro. See Chen et al., Plant Pathol.40:612-620 (1991), which reports that PAP has no effect on the growth ofthe fungi Phytophthora infestans, Colletotrichum coccodes, fusariumsolani, fusarium sulphureum, Phoma foreata and Rhizoctonia solani, invitro.

Hence, a need remains for a means by which to confer broad spectrumvirus and/or fungus resistance to plants without causing cell death orsterility, and which requires a minimum number of transgenes.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to PAP mutants having reducedphytotoxicity, and which exhibit PAP biological activity in plants. By"PAP biological activity," it is meant PAP anti-viral activity and/orPAP anti-fungal activity. One preferred group of PAP mutants ischaracterized by at least one amino acid substitution in the N-terminusof mature PAP, such as a substitution for the Glycine 75 residue or theGlutamic acid 97 residue. Another group of PAP mutants is characterizedby a truncation of as many as 38 amino acids at the N-terminus of maturePAP. Yet another preferred group of PAP mutants is characterized bymutations such as truncations in the C-terminal region of mature PAP.More preferred are PAP mutants truncated at their C-terminus by at leastabout 26 to about 76 mature PAP amino acids (not counting the 29-aminoacid C-terminal extension of wild-type PAP). A further group of PAPmutants are enzymatically inactive and do not exhibit PAP anti-viralactivity E vitro or in planta; yet, they exhibit PAP anti-fungalactivity in plants. The PAP mutants of the present invention may alsoinclude the 22-amino acid N-terminal signal sequence and/or theC-terminal extension of wild-type PAP.

The present invention also provides DNA molecules encoding the PAPmutants, which may or may not also encode the 22-amino acid N-terminalsignal sequence of mature PAP and/or the 29-amino acid C-terminalextension of wild-type PAP. The DNAs can be operably linked to apromoter functional in procaryotic cells (e.g., E. coli), or eukaryoticcells such as plants, and then stably transformed into a vectorfunctional in said cells. Hosts, e.g., procaryotic or eukaryotic cells(e.g., yeast or plants), stably transformed with a mutant PAP-encodingDNA are also provided, as well as protoplasts stably transformed withthe DNAs. Transgenic plants and seed containing the DNAs are alsoprovided. Expression of the DNAs in the transgenic plants confers broadspectrum virus and/or fungus resistance upon the plants without being asphytotoxic to the plant as wild-type PAP. Plants included within thescope of the present invention are monocots, such as cereal crops, anddicot plants.

The present invention further provides a method for identifying a PAPmutant having reduced phytotoxicity and which exhibits PAP biologicalactivity in plants. The method involves the steps of providing atransformed eukaryotic cell such as yeast containing a maturePAP-encoding DNA molecule operably linked to an inducible promoterfunctional in the eukaryotic cell. The PAP-encoding DNA is mutagenizedprior to transformation, or the transformed cell is mutagenized (i.e.,the mutagenesis is performed after the cell is transformed with the PAPconstruct). The thus-transformed cells are cultured in a suitablemedium, and after a predetermined time, an inducer is added to themedium to cause expression of the DNA molecule. A determination is thenmade as to whether the survival of cultured cells is due to theexpression of a mutant PAP. Such mutant PAPs which exhibit a substantiallack of toxicity to the host would be considered as PAP mutants whichexhibit reduced phytotoxicity. The thus-identified PAP mutants whichalso exhibit broad spectrum virus and/or fungus resistance, asdetermined by in vitro (e.g. by exogenous application of the virus orfungus), or in vivo (e.g., by expression in transgenic plants); wouldalso be considered as PAP mutants which retain PAP biological activityin plants. The present invention further provides isolated and purifiedPAP mutants identified by the aforesaid method.

DETAILED DESCRIPTION OF THE INVENTION

Transgenic plants expressing DNAs encoding the PAP mutants of thepresent invention exhibit reduced phytotoxicity compared to transgenicplants that produce mature, wild-type PAP, ("PAP"), or variant PAP, i.e.PAP-v, but also exhibit anti-viral and/or anti-fungal activities. By theterm "reduced phytotoxicity," it is meant that a transgenic plant whichexpresses a mutant PAP-encoding DNA exhibits a normal and fertilephenotype and does not exhibit the stunted, mottled phenotypecharacteristic of transgenic plants that produce mature PAP (asdisclosed in Lodge, supra., for example). By "wild-type PAP," it ismeant the PAP amino acid sequence 1-262, the 22-amino acid N-terminalsignal peptide ("the N-terminal signal sequence of wild-type PAP"), andthe 29 amino acid C-terminal extension (amino acids enumerated 263-291),all illustrated in Table 1 below as SEQ ID NO: 2. The correspondingnucleotide sequence is set forth as SEQ ID NO: 1. Thus, by the terms"wild-type, mature PAP," or "mature PAP", it is meant the PAP amino acidsequence 1-262 shown in Table I.

                                      TABLE 1                                     __________________________________________________________________________    5'CTATGAAGTCGGGTCAAAGCATATACAGGCTATGCATTGTTAGAAACATTGATGCCT                                                             (SEQ ID NO:2)                          - CTGATCCCGATAAACAATACAAATTAGACAATAAGATGACATACAAGTACCTAAACTG                  - TGTATGGGGGAGTGAAACCTCAGCTGCTAAAAAAACGTTGTAAGAAAAAAAGAAAGT                   - TGTGAGTTAACTACAGGGCGAAAGTATTGGAACT                                                                      A                                                AGCTAGTAGGAAGGGAAG ATG AAG TCG ATG CTT GTG GTG ACA ATA TCA ATA                                   Met Lys Ser Met Leu Val Val Thr Ile Ser Ile                                                           (67)                               TGG CTC ATT CTT GCA CCA ACT TCA ACT TGG GCT GTG AAT ACA ATC ATC TAC                                                    Trp Leu Ile Leu Ala Pro Thr                                                  Ser Thr Trp Ala Val Asn Thr Ile                                               Ile Tyr                                                                           (1)                                                  (100)                             G                        AAT GTT GGA AGT ACC ACC ATT AGC AAA TAC GCC ACT TTT CTG AAT GAT CTT                                                    Asn Val Gly Ser Thr Thr Ile                                                  Ser Lys Tyr Ala Thr Phe Leu Asn                                               Asp Leu                                          (10)                                     (20)                      CGT AAT GAA GCG AAA GAT CCA AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG                                                    Arg Asn Glu Ala Lys Asp Pro                                                  Ser Leu Lys Cys Tyr Gly Ile Pro                                               Met Leu                                                       (30)                                    (40)                                                   C                                    CCC AAT ACA AAT ACA AAT CCA AAG TAC GTG TTG GTT GAG CTC CAA GGT TCA                                                    Pro Asn Thr Asn Thr Asn Pro                                                  Lys Tyr Val Leu Val Glu Leu Gln                                               Gly Ser                                                                  (50)                                       AAT AAA AAA ACC ATC ACA CTA ATG CTG AGA CGA AAC AAT TTG TAT GTG ATG                                                    Asn Lys Lys Thr Ile Thr Leu                                                  Met Leu Arg Arg Asn Asn Leu Tyr                                               Val Met                                       (60)                                    (70)                          GGT TAT TCT GAT CCC TTT GAA ACC AAT AAA TGT CGT TAC CAT ATC TTT AAT                                                    Gly Tyr Ser Asp Pro Phe Glu                                                  Thr Asn Lys Cys Arg Tyr His Ile                                               Phe Asn                                                   (80)                                    (90)                                                       GAT ATC TCA GGT ACT GAA CGC                                                  CAA GAT GTA GAG ACT ACT CTT TGC                                               CCA AAT                               Asp Ile Ser Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Leu Cys Pro Asn                                                    (100)                                GCC AAT TCT CGT GTT AGT AAA AAC ATA AAC TTT GAT AGT CGA TAT CCA ACA                                                    Ala Asn Ser Arg Val Ser Lys                                                  Asn Ile Asn Phe Asp Ser Arg Tyr                                               Pro Thr                                   (110)                                   (120)                             TTG GAA TCA AAA GCG GGA GTA AAA TCA AGA AGT CAG GTC CAA CTG GGA ATT                                                    Leu Glu Ser Lys Ala Gly Val                                                  Lys Ser Arg Ser Glu Val Gln Leu                                               Gly Ile                                               (130)                                  (140)                  CAA ATA CTC GAC AGT AAT ATT GGA AAG ATT TCT GGA GTG ATG TCA TTC ACT                                                    Gln Ile Leu Asp Ser Asn Ile                                                  Gly Lys Ile Ser Gly Val Met Ser                                               Phe Thr                                                          (150)                                              GAG AAA ACC GAA GCC GAA TTC CTA TTG GTA GCC ATA CAA ATG GTA TCA GAG                                                    Glu Lys Thr Glu Ala Glu Phe                                                  Leu Leu Val Ala Ile Gln Met Val                                               Ser Glu                               (160)                                  (170)                                  GCA GCA AGA TTC AAG TAC ATA GAG AAT CAG GTG AAA ACT AAT TTT AAC AGA                                                    Ala Ala Arg Phe Lys Tyr Ile                                                  Glu Asn Gln Val Lys Thr Asn Phe                                               Asn Arg                                           (180)                                   (190)                     GCA TTC AAC CCT AAT CCC AAA 6TA CTT AAT TTG CAA GAG ACA TGG GGT AAG                                                    Ala Phe Asn Pro Asn Pro Lys                                                  Val Leu Asn Leu Gln Glu Thr Trp                                               Gly Lys                                                       (200)                                  (210)                                                   ATT TCA ACA GCA ATT CAT GAT                                                  GCC AAG AAT GGA GTT TTA CCC AAA                                               CCT CTC                               Ile Ser Thr Ala Ile His Asp Ala Lys Asn Gly Val Leu Pro Lys Pro Leu                                                       (220)                             GAG CTA GTG GAT GCC AGT GGT GCC AAG TGG ATA GTG TTC AGA GTG GAT GAA                                                    Glu Leu Val Asp Ala Ser Gly                                                  Ala Lys Trp Ile Val Leu Arg Val                                               Asp Glu                                      (230)                                   (240)                          ATC AAG CCT GAT GTA GCA CTC TTA AAC TAC GTT GGT GGG AGC TGT CAG ACA                                                    Ile Lys Pro Asp Val Ala Leu                                                  Leu Asn Tyr Val Gly Gly Ser Cys                                               Gln Thr                                                   (250)                                  (260)                                                       ACT TAT AAC CAA AAT GCC ATG                                                  TTT CCT CAA CTT ATA ATG TCT ACT                                               TAT TAT                               Thr Tyr Asn Gln Asn Ala Met Phe Pro Gln Leu Ile Met Ser Thr Tyr Tyr                                                    (262)                                                                        (270)                                 AAT TAC ATG GTT AAT CTT GGT GAT CTA TTT GAA GGA TTC TGATCATAAACA                                                       Asn Tyr Met Val Asn Leu Gly                                                  Asp Leu Phe Glu Gly Phe                                                            (280)                                                                                 (290)                    TAATAAGGAGTATATATATATTACTCCAACTATATTATAAAGCTTAAATAAGAGGCCG (SEQ ID                                                    NO.1)                                  - TGTTAATTAGTACTTGTTGCCTTTTGCTTTATGGTGTTGTTTATTATGCCTTGTATGCTT                                                         - GTAATATTATCTAGAGAACAAGATGTAC                                              TGTGTAATAGTCTTGTTTGAAATAAAACTT                                                  - CCAATTATGATGCAAAAAAAAAAAAAAA                                              3'                                  __________________________________________________________________________

Table I further shows PAP-v amino acids and corresponding nucleotides inproper alignment with wild-type PAP. Basically, the amino acid sequenceof PAP-v differs from that of wild-type PAP in terms of a Leu20Arg(i.e., an arginine residue at position 20 of mature PAP as opposed to aleucine residue) and a Tyr49His substitution. The third change in thePAP-v nucleotide sequence (TCG→TCA codon for the first occurring Ser inthe signal sequence) has no effect on the amino acid sequence. Thus, thecorresponding PAP-v nucleotide and amino acid sequences are representedas follows:

                                      TABLE II                                    __________________________________________________________________________      5'CTATGAAGTCGGGTCAAAGCATATACAGGCTATGCATTGTTAGAAACATTGATGCCTCTGATC            CCGATAAACAATACAAATTAGACAATAAGATGACATACAAGTACCTAAACTGTGTATGGGGGA               GTGAAACCTCAGCTGCTAAAAAAACGTTGTAAGAAAAAAAGAAAGTTGTGAGTTAACTACAGG               GCGAAAGTATTGGAACT                                                              - AGCTAGTAGGAAGGGAAG ATG AAG TCA ATG CTT GTG GTG ACA ATA TCA ATA                                Met Lys Ser Met Leu Val Val Thr Ile Ser Ile                 -                                         (67)                               TGG CTC ATT CTT GCA CCA ACT TCA ACT TGG GCT GTG AAT ACA ATC ATC TAC           Trp Leu Ile Leu Ala Pro Thr Ser Thr Trp Ala Val Asn Thr Ile Ile Tyr                                                       (1)                                -                 (100)                                                      AAT GTT GGA AGT ACC ACC ATT AGC AAA TAC GCC ACT TTT CGG AAT GAT CTT           Asn Val Gly Ser Thr Thr Ole Ser Lys Tyr Ala Thr Phe Arg Asn Asp Leu                       (10)                                    (20)                       - CGT AAT GAA GCG AAA GAT CCA AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG        Arg Asn Glu Ala Lys Asp Pro Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu                                   (30)                                    (40)           - CCC AAT ACA AAT ACA AAT CCA AAG CAC GTG TTG GTT GAG CTC CAA GGT TCA        Pro Asn Thr Asn Thr Asn Pro Lys His Val Leu Val Glu Leu Gln Gly Ser                                               (50)                                       - AAT AAA AAA ACC ATC ACA CTA ATG CTG AGA CGA AAC AAT TTG TAT GTG ATG        Asn Lys Lys Thr Ile Thr Leu Met Leu Arg Arg Asn Asn Leu Tyr Val Met                   (60)                                    (70)                           - GGT TAT TCT GAT CCC TTT GAA ACC AAT AAA TGT CGT TAC CAT ATC TTT AAT        Gly Tyr Ser Asp Pro Phe Glu Thr Asn Lys Cys Arg Tyr His Ile Phe Asn                               (80)                                    (90)               - GAT ATC TCA GGT ACT GAA CGC CAA GAT GTA GAG ACT ACT CTT TGC CCA AAT        Asp Ile Ser Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Leu Cys Pro Asn                                           (100)                                          - GCC AAT TCT CGT GTT AGT AAA AAC ATA AAC TTT GAT AGT CGA TAT CCA ACA        Ala Asn Ser Arg Val Ser Lys Asn Ile Asn Phe Asp Ser Arg Tyr Pro Thr               (110)                                   (120)                              - TTG GAA TCA AAA GCG GGA GTA AAA TCA AGA AGT CAG GTC CAA CTG GGA ATT        Leu Glu Ser Lys Ala Gly Val Lys Ser Arg Ser Gln Val Gln Leu Gly Ile                           (130)                                   (140)                  - CAA ATA CTC GAC AGT AAT ATT GGA AAG ATT TCT GGA GTG ATG TCA TTC ACT        Gln Ile Leu Asp Ser Asn Ile Gly Lys Ile Ser Gly Val Met Ser Phe Thr                                       (150)                                              - GAG AAA ACC GAA GCC GAA TTC CTA TTG GTA GCC ATA CAA ATG GTA TCA GAG        Glu Lys Thr Glu ala Glu Phe Leu Leu Val Ala Ile Gln Met Val Ser Glu           (160)                                   (170)                                  - GCA GCA AGA TTC AAG TAC ATA GAG AAT CAG GTG AAA ACT AAT TTT AAC AGA        Ala Ala Arg Phe Lys Tyr Ile Glu Asn Gln Val Lys Thr Asn Phe Asn Arg                       (180)                                   (190)                      - GCA TTC AAC CCT AAT CCC AAA GTA CTT AAT TTG CAA GAG ACA TGG GGT AAG        Ala Phe Asn Pro Asn Pro Lys Val Leu Asn Leu Gln Glu Thr Trp Gly Lys                                   (200)                                   (210)          - ATT TCA ACA GCA ATT CAT GAT GCC AAG AAT GGA GTT TTA CCC AAA CCT CTC        Ile Ser Thr Ala Ile His Asp Ala Lys Asn Gly Val Leu Pro Lys Pro Leu                                           (220)                                          - GAG CTA GTG GAT GCC AGT GGT GCC AAG TGG ATA GTG TTG AGA GTG GAT GAA        Glu Leu Val Asp Ala Ser Gly Ala Lys Trp Ile Val Leu Arg Val Asp Glu                   (230)                                   (240)                          - ATC AAG CCT GAT GTA GCA CTC TTA AAC TAC GTT GGT GGG AGC TGT CAG ACA -     Ile Lys Pro Asp Val Ala Leu Leu Asn Tyr Val Gly Gly Ser Cys Gln Thr                                (250)                                   (260)              - ACT TAT AAC CAA AAT GCC ATG TTT CCT CAA CTT ATA ATG TCT ACT TAT TAT        Thr Tyr Asn Gln Asn Ala Met Phe Pro Gln Leu Ile Met Ser Thr Tyr Tyr           (262)                           (270)                                          - AAT TAC ATG GTT AAT CTT GGT GAT CTA TTT GAA GGA TTC TGATCATAAACA           Asn Tyr Met Val Asn Leu Gly Asp Leu Phe Glu Gly Phe (SEQ ID NO: 4)                (280)                                   (290)                              - TAATAAGGAGTATATATATATTACTCCAACTATATTATAAAGCTTAAATAAGAGGCCGTGTTAA           TTAGTACTTGTTGCCTTTTGCTTTATGGTGTTGTTTATTATGCCTTGTATGCTTGTAATATTATCT            AGAGAACAAGATGTACTGTGTAATAGTCTTGTTTGAAATAAAACTTCCAATTATGATGCAAAAA              AAAAAAAAAA3'(SEQ ID NO: 3)                                                   __________________________________________________________________________

Tables I and II also show 5' and 3' non-coding, flanking sequences.

Upon expression in eukaryotic cells, the N-terminal 22-amino acidsequence of wild-type PAP is co-translationally cleaved, yielding apolypeptide having a molecular weight of about 32 kD, which is thenfurther processed by the cleavage of the C-terminal 29-amino acids ("theC-terminal extension of wild-type PAP" or "PAP (263-292)"), yieldingmature, wild-type PAP (hereinafter "PAP (1-262)") (i.e., that which isisolated from Phytolacca americana leaves), having a molecular weight ofabout 29 kD. See Irvin et al., Pharmac. Ther. 55:279-302 (1992); Dore etal., Nuc. Acids Res. 21(18):4200-05 (1993); Monzingo et al., J. Mol.Biol. 2:705-15 (1993); Tumer et al., Proc. Natl. Acad. Sci. USA92:8448-8452 (1995).

By the phrase "PAP anti-viral activity," it is meant that the expressionof a mutant PAP of the present invention in a transgenic plant confersbroad spectrum virus resistance, i.e., resistance to or the capabilityof suppressing infection by a number of unrelated viruses, including butnot limited to RNA viruses e.g., potexviruses such as (PVX, potato virusX), potyvirus (PVY), cucumber mosaic virus (CMV), tobacco mosaic virusesUI), barley yellow dwarf virus (BYDV), wheat streak mosaic virus, potatoleaf roll virus (PLRV), plumpox virus, watermelon mosaic virus, zucchiniyellow mosaic virus, t papaya ringspot virus, beet western yellow virus,soybean dwarf virus, carrot read leaf virus and DNA plant viruses suchas tomato yellow leaf curl virus. See also Lodge et al., su ., Tomlinsonet al., J. Gen. Virol. 22:225-32 (1974); and Chen et al., Plant Pathol.40:612-20 (1991).

By the phrase "PAP anti-fungal activity", it is meant that the mutantPAPs of the present invention confer broad spectrum fungal resistance toplants. The mutant PAPs of the present invention provide increasedresistance to diseases caused by plant fungi, including those caused byPythium (one of the causes of seed rot, seedling damping off and rootrot), Phytophthora (the cause of late blight of potato and of root rots,and blights of many other plants), Bremia, Peronospora, Plasmopara,Pseudoperonospora and Sclerospora (causing downy mildews), Erysiphegraminis (causing powdery mildew of cereals and grasses), Verticillium(causing vascular wilts of vegetables, flowers, crop plants and trees),Rhizoctonia (causing damping off disease of many plants and brown patchdisease of turfgrasses), Fusarium (causing root rot of bean, dry rot ofpotatoes), Cochliobolus (causing root and foot rot, and also blight ofcereals and grasses), Giberella (causing seedling blight and foot orstalk rot of corn and small grains), Gaeumannomyces (causing thetake-all and whiteheads disease of cereals), Schierotinia (causing crownrots and blights of flowers and vegetables and dollar spot disease ofturfgrasses), Puccinia (causing the stem rust of wheat and other smallgrains), Ustilago (causing corn smut), magnaporthae (causing summerpatch of turfgrasses), and Schierotium (causing southern blight ofturfgrasses). Other important fungal diseases include those caused byCercospora, Septoda, Mycosphoerella, Glomerella, Colletotrichum,Helminthosporium, Alterneria, Botrytis, Cladospodum, and Aspergillus.

Applicant also believes that the mutant PAPs of the present inventionconfer increased resistance to insects, bacteria and nematodes inplants. Important bacterial diseases include those caused byPseudomonas, Xanthomonas, Erwinia, Clavibacter and Streptomyces.

The PAP mutants of the present invention differ from wild-type PAPsubstantially as follows: (1) those which exhibit alteredcompartmentalization in vivo; (2) C-terminal mutants, including but notlimited to deletion or frameshift mutants; (3) N-terminal mutants; and(4) active-site mutants. The first category of PAP mutants may havealtered compartmentalization properties in vivo; that is, they may notbe localized in the same subcellular compartment as wild-type PAP. Whilenot intending to be bound to any particular theory of operation,Applicant believes that these PAP mutants are unable to undergoco-translational processing (to remove the 22 amino acid signal peptide)and/or post-translational processing (to remove the 29-amino acidC-terminal fragment) in yeast, which results in substantially diminishedor negligible cytotoxicity. These mutants are also non-phytotoxic. Whatis particularly surprising or unexpected about the function of thesemutant PAPs in vivo is that the mutations are located within thesequence encoding the mature PAP (1-262), and not within the N-terminalsignal peptide or the 29-amino acid C-terminal extension. In addition,the mutant PAPs are enzymatically active in inhibiting translation invitro, indicating that phytotoxicity is not solely a function ofenzymatic activity. Preferred PAP mutants include a conservative pointmutation such that wild-type PAP amino acid residue 75 glycine (Gly75)is changed to valine, alanine, isoleucine or leucine, or (2) aconservative or non-conservative point mutation at wild-type PAP aminoacid residue 97 Glutamic acid (Glu97). More preferred PAP mutants arePAP (1-262, Gly75Val) and PAP (1-262, Glu97Lys), the respective DNAs ofwhich can be prepared simply by changing the wild-type GGT codon forglycine75 to GfT (valine), and the GAA codon for glutamic acid 97 to AAA(lysine). Other PAP mutants having altered compartmentalizationproperties can be identified by the selection method described below.Dore et al., sa, disclose an Arg67Gly PAP mutant (numbered in D as Arg68Gly due to the presence of an N-terminal methionine residue), butwhich is toxic to eukaryotic cells and non-toxic to procaryotic cellssuch as E. on. This mutant is not included within the scope of thepresent invention.

The second category of PAP mutants of the present invention havedeletions or amino acid substitutions in the C-terminal region of PAP.Applicant has unexpectedly discovered that these mutants are alsonon-toxic in vivo (i.e., non-phytotoxic) even though they inhibittranslation in vitro. Preferred mutants have deletions of from about 26to about 76 amino acids of mature PAP, and more preferred are the PAPmutants PAP (1-236)-PAP (1-184), inclusive. Thus, truncations beginningat about amino acid residue 237 of wild-type mature PAP, e.g., PAP(1-236), PAP (1-235), PAP (1-234), PAP (1-233), PAP (1-232), PAP(1-231), PAP (1-230), PAP (1-229), PAP (1-228), PAP (1-227), PAP(1-226), PAP (1-225), PAP (1-224), PAP (1-223), PAP (1-222), PAP(1-221), PAP (1-220), PAP (1-219), PAP (1-218), PAP (1-217), PAP(1-216), PAP (1-215), PAP (1-214), PAP (1-213), PAP (1-212), PAP(1-211), PAP (1-210), PAP (1-209), PAP (1-208), PAP (1-207), PAP(1-206), PAP (1-205), PAP (1-204), PAP (1-203), PAP (1-202), PAP(1-201), PAP (1-200), PAP (1-199), PAP (1-198), PAP (1-197), PAP(1-196), PAP (1-195), PAP (1-194), PAP (1-193), PAP (1-192), PAP(1-191), PAP (1-190), PAP (1-189), PAP (1-188), PAP (1-187), PAP(1-186), PAP (1-185), and PAP (1-184) are encompassed by the presentinvention. More preferred mutants include PAP (1-184Glu), PAP(1-188Lys), PAP (1-206Glu), PAP (1-209) and PAP (1-236Lys). Deletionsshorter than about 26 (i.e., between 1 and 25 amino acids, inclusive) orlonger than 76 mature PAP amino acids are included in the scope of thepresent invention provided that they are non-toxic to plant cells, whichcan be determined by the selection method described in detail below, andthey confer fungus and/or virus resistance in planta. The latterproperties can be determined in vitro, e.g. by inoculating plant parts,e.g. leaves, with the PAP mutant in the presence of a virus or fungus,or by separate in vivo assays wherein a transgenic plant transformedwith a mutant PAP-encoding DNA is inoculated with a fungus or virus. Apreferred C-terminal substitution mutant is PAP (1-262, Leu202Phe).Again, while not intending to be bound by any particular theory ofoperation, Applicant believes that the sequence of PAP amino acids244Glu-259Cys (shown in Table I), which is homologous to the consensussequence for the prokaryotic membrane lipoprotein lipid attachment site(Hayashi et al., J. Bioenerg. Biomem. 22:451-71 (1990)), and which isabsent from each of the PAP mutants disclosed above, is involved inbinding of PAP to phospholipids on endoplasmic reticulum (ER) membraneswhich facilitates the translocation of PAP into the cytosol of the cellwhere it inhibits protein synthesis. Disarming this function, e.g., bydeletion or by frameshift mutation, results in PAP mutants having theinstantly disclosed properties. o et al., supra, also disclose aPhe195Tyr, Lys211Arg PAP mutant (which numbering is +1 out-of-phase withthe numbering used herein due to the N-terminal Met residue required forexpression in E. coli), which is toxic to eukaryotic cells (such asplants) but non-toxic to procaryotes such as E. coli. Accordingly, thisPAP mutant disclosed in the D=publication is not included within thescope of the present invention.

The third category of PAP mutants is characterized by truncations offrom 1 to at least about 38 N-terminal amino acid residues of maturePAP. These mutants include PAP (2-262), PAP (3-262), PAP (4-262), PAP(5-262), PAP (6-262), PAP (7-262), PAP (8-262), PAP (9-262), PAP(10-262), PAP (11-262), PAP (12-262), PAP (13-262), PAP (14-262), PAP(15-262), PAP (16-262), PAP (17-262), PAP (18-262), PAP (19-262), PAP(20-262), PAP (21-262), PAP (22-262), PAP (23-262), PAP (24-262), PAP(25-262), PAP (26-262), PAP (27-262), PAP (28-262), PAP (29-262), PAP(30-262), PAP (31-262), PAP (32-262), PAP (33-262), PAP (34-262), PAP(35-262), PAP (36-262), PAP (37-262), PAP (38-262) and PAP (39-262).Truncations of greater than 38 N-terminal amino acid residues of maturePAP are included within the scope of the present invention to the extentthat they exhibit PAP biological activity and reduced phytotoxicity invivo. These properties may be determined in accordance with theprocedures set forth in the working examples, below.

The fourth category of PAP mutants contain active-site mutations whichrender the PAP molecule enzymatically inactive (as measured by theirlack of ability to inhibit translation in vitro and/or in eukaryoticribosomes). Applicant has surprisingly and unexpectedly found that thesemutants exhibit broad spectrum anti-fungal activity when expressed inplants, even though they exhibit negligible PAP anti-viral activity. Theputative active site of PAP includes amino acid residues Tyr72, Tyr 123,Glul76, Arg179 and Trp208. Accordingly, PAP active site mutants, e.g.,which contain a conservative or even non-conservative substitution inthe PAP active site or wherein at least one active site amino acid isdeleted or replaced by another amino acid, wherein the PAP is renderedenzymatically inactive but retains anti-fungal activity, are encompassedwithin the present invention. PAP mutants can be tested for enzymaticand anti-fungal activities using the assay procedures described in theworking examples, below. A preferred PAP active site mutant is PAP(1-262, Glu176Val).

In regard to the disclosed PAP mutants, the phrase "which differs fromwild-type PAP substantially in that . . . " means that except for theamino acid changes (described above), that are necessary to conferreduced phytotoxicity and anti-viral and/or anti-fungal activity, theamino acid sequences of the mutant PAPs are substantially identical tothat of mature PAP. By the term "substantially identical," it is meantthat the PAP mutants of the present invention can be further modified byway of additional substitutions, additions or deletions provided thatthe resultant PAP mutant retains reduced phytotoxicity and PAPbiological activity as defined herein. For example, the N-terminus ofthe mutant PAP may be changed to a methionine residue, either bysubstitution or addition, to allow for expression of a DNA encoding themutant PAP in various host cells particularly E. coli. The PAP mutantsof the present invention may further include the N-terminal 22-aminoacid signal peptide of wild-type PAP and/or the 29-amino acid C-terminalextension, both of which are shown in Table I above.

DNAs encoding the mutant PAPs of the present invention can be preparedby manipulation of known PAP genes. See Ausubel et al. (eds.), Vol. 1,Chap. 8 in Current Protocols in Molecular Biology, Wiley, N.Y. (1990).The DNAs may also be prepared via PCR techniques. See PCR Protocols,Innis et al. (eds.), Academic Press, San Diego, Calif. (1990). Themutant PAP-encoding DNA (e.g., a cDNA) is preferably inserted into aplant transformation vector in the form of an expression cassettecontaining all of the necessary elements for transformation of plantcells. The expression cassette typically contains, in proper readingframe, a promoter functional in plant cells, a 5' non-translated leadersequence, the mutant PAP DNA, and a 3' non-translated region functionalin plants to cause the addition of polyadenylated nucleotides to the 3'end of the RNA sequence. Promoters functional in plant cells may beobtained from a variety of sources such as plants or plant DNA viruses.The selection of a promoter used in expression cassettes will determinethe spatial and temporal expression pattern of the construction in thetransgenic plant. Selected promoters may have constitutive activity andthese include the CaMV 35S promoter, the actin promoter (McElroy et al.Plant Cell 2:163-71 (1990); McElroy et al. Mol. Gen. Genet. 231:150-160(1991); Chibbar et al. Plant Cell Rep. 12:506-509 (1993), and theubiquitin promoter (Binet et al. Plant Science 79:87-94 (1991),Christensen et al. Plant Mol. Biol. 12:619-632 (1989); Taylor et al.Plant Cell Rep. 12:491-495 (1993)). Alternatively, they may bewound-induced (Xu et al., Plant Mol. Biol 22:573-588 (1993), Logemann etal., Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Mol. Bio.22:783-792 (1993), Firek et al. Plant Mol. Biol. 22:129-142 (1993),Warener et al. Plant J. 3:191-201 (1993)) and thus drive the expressionof the mutant PAP gene at the sites of wounding or pathogen infection.Other useful promoters are expressed in specific cell types (such asleaf epidermal cells, meosphyll cells, root cortex cells) or in specifictissues or organs (roots, leaves or flowers, for example). PatentApplication WO 93/07278, for example, describes the isolation of themaize tnpA gene which is preferentially expressed in pith cells.Hudspeth and Grula, Plant Mol. Biol. 12:579-589 (1989), have described apromoter derived from the maize gene encoding phosphoenolpyruvatecarboxylase (PEPC) with directs expression in a leaf-specific manner.Alternatively, the selected promoter may drive expression of the geneunder a light-induced or other temporally-regulated promoter. A furtheralternative is that the selected promoter be chemically regulated. TheDNAs may also encode the N-terminal signal sequence and/or theC-terminal extension of wild-type PAP.

A variety of transcriptional cleavage and polyadenylation sites areavailable for use in expression cassettes. These are responsible forcorrect processing (formation) of the 3' end of mRNAs. Appropriatetranscriptional cleavage and polyadenylation sites which are known tofunction in plants include the CaMV 35S cleavage and polyadenylationsites, the tml cleavage and polyadenylations sites, the nopalinesynthase cleavage and polyadenylation sites, the pea rbcS E9 cleavageand polyadenylation sites. These can be used in both monocotyledons anddicotyledons.

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants. Various intron sequences have beenshown to enhance expression, particularly in monocotyledonous cells. Forexample, the introns of the maize Adh1 gene have been found tosignificantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells. Intron 1 was found tobe particularly effective and enhanced expression in fusion constructswith the chloramphenicol acetyltransferase gene (Callis et al., GenesDevelop 1:1183-1200 (1987)). In the same experimental system, the intronfrom the maize bronze-l gene had a similar effect in enhancingexpression (Callis et al., supra.). Intron sequences have been routinelyincorporated into plant transformation vectors, typically within thenon-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the "Ω-sequence"), Maize Chlorotic mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 1:8693-8711(1987); Skuzeski et al. Plant Mol. Biol. 1:65-79 (1990)).

Numerous transformation vectors are available for plant transformation,and the genes of this invention can be used in conjunction with any suchvectors. The selection of vector for use will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformations include the nptII gene which confers resistance tokanamycin (Messing and Vierra, Gene 1:259-268 (1982); Bevan et al.,Nature 304:184-187 (1983)), the bar gene which confers resistance to theherbicide phosphinothricin (White et al., Nucl. Acids Res. 18:1062(1990); Spencer et al., Theor. Appl. Genet. 22:625-631 (1990)), the hphgene which confers resistance to the antibiotic hygromycin (Blochingerand Diggelmann, Mol. Cell Biol. 4:2929-2931)), and the dhfr gene, whichconfers resistance to methotrexate (Fling & Elwell, 1980)). Vectorssuitable for Agrobacterium transformation typically carry at least oneT-DNA border sequence. These include vectors such as pBIN19 (Bevan,Nucl. Acids Res. (1984) and pCIB200 (EP 0 332 104).

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques which do not rely on Agrobacteriwninclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. For example, pCIB3064 is a pUC-derived vector suitable forthe direct gene transfer technique in combination with selection by theherbicide basta (or phosphinothricin). It is described in WO 93/07278and Koziel et al. (Biotechnology 11:194-200 (1993)).

An expression cassette containing the mutant PAP gene DNA containing thevarious elements described above may be inserted into a planttransformation vector by standard recombinant DNA methods.Alternatively, some or all of the elements of the expression cassettemay be present in the vector, and any remaining elements may be added tothe vector as necessary.

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterum-based techniques and techniques which do notrequire Agrobacteriwn. Non-Agrobacteyium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described in Paszkowski et al., EMBO J 3:2717-2722(1984), Potrykis et al., Mol. Gen. Genet. 122:169-177 (1985), Reich etal., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species which are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432(tomato), WO 87107299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)).Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend on thecomplement of Wir genes carried by the host Agrobacterium strain eitheron a co-resident plasmid or chromosomally (e.g. strain CIB542 forpCIB200 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of therecombinant binary vector, to Agrobacterium is accomplished by atriparental mating procedure using E. coil carrying the recombinantbinary vector, a helper E. coli strain which carries a plasmid such aspRK2013 which is able to mobilize the recombinant binary vector to thetarget Agrobacterium strain. Alternatively, the recombinant binaryvector can be transferred to Agrobacterium by DNA transformation (Hofgen& Willmitzer, Nucl. Acids Res. 1:9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols known in the art. Transformed tissue isregenerated on selectable medium carrying an antibiotic or herbicideresistance marker present between the binary plasmid T-DNA borders.

Preferred transformation techniques for monocots include direct genetransfer into protoplasts using PEG or electroporation techniques andparticle bombardment into callus tissue. Transformation can beundertaken with a single DNA species or multiple DNA species (i.e.co-transformation) and both these techniques are suitable for use withthis invention. Co-transformation may have the advantage of avoidingcomplex vector construction and of generating transgenic plants withunlinked loci for the gene of interest and the selectable marker,enabling the removal of the selectable marker in subsequent generations,should this be regarded desirable. However, a disadvantage of the use ofco-transformation is the less than 100% frequency with which separateDNA species are integrated into the genome (Schocher et al.,Biotechnology 4:1093-1096 (1986)).

Published Patent Applications EP 0 292 435, EP 0 392 225 and WO 93/07278describe techniques for the preparation of callus and protoplasts ofmaize, transformation of protoplasts using PEG or electroporation, andthe regeneration of maize plants from transformed protoplasts.Gordeon-Kamm et al., Plant Cell 2:603-618 (1990), and Fromm et al.,Biotechnology 11:194-200 (1993), describe techniques for thetransformation of elite inbred lines of maize by particle bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhange et al., Plant Cell Rep. 7:739-384 (1988);Shimamoto et al. Nature 338:274-277 (1989); Datta et al. Biotechnology8:736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 2:957-962 (1991)).

Patent Application EP 0 332 581 described techniques for the generation,transformation and regeneration of Pooideae protoplasts. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10:667-674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102:1077-1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus.

Transformation of monocot cells such as Zea mays can be achieved bybringing the monocot cells into contact with a multiplicity ofneedle-like bodies on which these cells may be impaled, causing arupture in the cell wall thereby allowing entry of transforming DNA intothe cells. See U.S. Pat. No. 5,302,523. Transformation techniquesapplicable to both monocots and dicots are also disclosed in thefollowing U.S. Pat. No. 5,240,855 (particle gun); U.S. Pat. No.5,204,253 (cold gas shock accelerated microprojectiles); U.S. Pat. No.5,179,022 (biolistic apparatus); U.S. Pat. Nos. 4,743,548 and 5,114,854(microinjection); U.S. Pat. Nos. 5,149,655, 5,120,657 (acceleratedparticle mediated transformation); U.S. Pat. No. 5,066,587 (gas drivenmicroprojectile accelerator); U.S. Pat. No. 5,015,580 (particle-mediatedtransformation of soy bean plants); U.S. Pat. No. 5,013,660 (laserbeam-mediated transformation);U.S. Pat. Nos. 4,849,355 and 4,663,292.

The thus-transformed plant cells or plant tissue are then grown intofull plants in accordance with standard techniques. Transgenic seed canbe obtained from transgenic flowering plants in accordance with standardtechniques. Likewise, non-flowering plants such as potato and sugarbeets can be propagated by a variety of known procedures. See, e.g.,Newell et al. Plant Cell Rep. 10:30-34 (1991) (disclosing potatotransformation by stem culture).

The mutant PAP encoding DNAs of the present invention confer broadspectrum fungus and virus resistance to any plant capable of expressingthe DNAs, including monocots (e.g., cereal crops) and dicots. Specificexamples include maize, tomato, turfgrass, asparagus, papaya, sunflower,rye, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, malt,wheat, alfalfa, soybean, oat, eggplant, squash, onion, broccoli,sugarcane, sugar beet, beets, apples, oranges, grapefruit, pear, plum,peach, pineapple, grape, rose, carnation, daisy, tulip, Douglas fir,cedar, white pine, scotch pine, spruce, peas, cotton, flax and coffee.

PAP mutants other than those specifically described above can beidentified by a selection system in eukaryotic cells. In a preferredembodiment, a PAP-encoding DNA molecule, operably linked to an induciblepromoter functional in the eukaryotic cell, is randomly mutagenized inaccordance with standard techniques. The cell is then transformed withthe mutagenized PAP construct. The thus-transformed cell is thencultured in a suitable medium for a predetermined amount of time, e.g.,sufficient to cause some growth of the cells, at which time an induceris added to the medium to cause expression of the mutagenized DNAmolecule. If the cultured cell survives the induction of the expressionof the mutagenized PAP DNA molecule, which indicates that themutagenesis resulted in the expression of a non-toxic PAP mutant, thePAP mutant can be then assayed in vitro or in vivo to determine whetherit retains PAP biological activity. Preferred in vitro assays includeeukaryotic translation systems such as reticulocyte lysate systemswherein the extent of the inhibition of protein synthesis in the systemcaused by the PAP mutant is determined. Preferred host cells are yeastcells such as Saccharomyces cerevisiae, as described in greater detailin Example 1, below. This method can also be conducted with a pluralityof randomly mutagenized PAP-encoding DNA molecules. The PAP mutantsidentified as having reduced phytotoxicity and which retain PAPanti-viral and/or anti-fungal activity, as determined by subsequentassays, can then be isolated, purified and sequenced in accordance withstandard techniques.

In another embodiment, the mutagenesis is performed after thetransformation of the eukaryotic cell. The disadvantage withmutagenizing the DNA after transformation is that the chromosomal DNA ofthe host can also be mutagenized. To determine whether the mutations ofthe surviving cells are chromosomal or plasmid-borne in nature, thisembodiment requires the step of replacing the transforming PAP-encodingDNA with wild-type PAP-encoding DNA under the control of an induciblepromoter, and growing the cells in the presence of the inducer. Mutantswhich retain the ability to grow are chromosomal mutants, whereasmutants which fail to grow are plasmid-borne (i.e., PAP) mutants.

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLE 1

A. Construction of yeast expression vectors and analysis of PAPexpression in yeast. The full length cDNAs corresponding to PAP andPAP-v disclosed in Lodge et al., and shown in Table 1, were cloned intoyeast expression vectors, under the control of the galactose induciblepromoter, GAL1. S. cerevisiae was chosen as the expression systembecause yeast has the advantage of supplying eukaryotic cell-specificpost-translational modifications. Since yeast ribosomes are sensitive toPAP, a regulated promoter was used to drive the expression of PAP. ThecDNAs encoding PAP and PAP-v were cloned into the yeast expressionvector pAC55, containing the selectable marker, URA3, as BgIII/SmaIfragments under the control of the galactose inducible promoter pGal1.The vectors containing PAP (NT123) and PAP-v (NT124) were transformedinto the yeast strain W303 (Mat a, ade2-1 trp1-1 ura3-1 leu2-3, 112his3-11, 15can1-100 ) (Bossie et al., Mol. Biol. Cell, 3:875-893(1992)), according to the procedure described in Ito et al., J.Bacteriol. 1:163-168 (1983), and transformants were selected on uracilminus medium with glucose at 30° C.

Yeast cells containing either NT123 (wild-type PAP) or NT124 (PAP-v)were grown in uracil minus medium with 2% raffinose at 30° C. for 48 hto a density of 5×10⁷ cells/ml. PAP protein expression was induced bythe addition of 2% galactose to half of the culture, while the otherhalf of the culture was used as an uninduced control. The cells wereallowed to grow for an additional 4 h and then collected bycentrifugation at 10,000 g for 5 min. The cells were resuspended in RIPAbuffer 9150 mM NaCl, 1% NPA40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mMTris-HCl, pH 8) with protease inhibitors (0.1 μg/ml each of antipain,aprotinin, chymostatin, leupeptin, pepstatin) and lysed using glassbeads (0.5 mm). Extracts were loaded on a 10% SDS-PAGE gel in accordancewith the procedures described in Tomlinson, J. Gen. Virol., supra.Immunoblot analysis was performed by the enhanced chemiluminescence(ECL) method (Amersham), using antibodies against purified PAP.

Both PAP and PAP-v were expressed in yeast after galactose induction.Based on comparison with PAP protein standards, yeast cells containingthe PAP plasmid (NT123) expressed both the mature form of PAP and alarger form, while cells containing the PAP-v (NT124) expressedpredominantly the larger form and very low levels of the mature form.PAP was not detected in the culture medium. While not intending to bebound by any particular theory of operation, Applicant believes thatthese results suggest the following: (1) PAP is expressed as a precursorand processed to the mature form in yeast; and (2) PAP undergoes furtherprocessing in addition to the co-translational cleavage of the aminoacid N-terminal signal peptide (Lodge et al., supra.) because the sizeof the mature PAP and the PAP expressed in yeast was found to be smallerthan the expected size after the removal of the signal sequence.

B. In vitro translation and processing of PAP and PAP-v. To examine theprocessing of PAP in vitro, both constructs described in Example 1A weretranscribed and translated in vitro using the T7 coupled reticulocytelysate translation system in the presence of 35S-methionine with orwithout canine microsomal membranes (Promega). PAP and PAP-v cDNAs werecloned into the pGem 3Z vector (Promega) downstream of the T7 promoter.An equal amount of DNA (1 μg) from each construct was transcribed andtranslated in vitro in the presence ³⁵ S-methionine, using the T7coupled reticulocyte lysate translation system (Promega) with or withoutcanine pancreatic microsomal membranes (Promega). Translation productswere incubated with 0.2 mg/ml proteinase K in the presence of 5 mM EDTAand 125 mM sucrose for 90 min. Proteinase K was inactivated by additionof 4mM PMSF and incubation at room temperature for 2 hours. Translationproducts were then treated with Endo-H (endo-N-acetylglucosaminidase) (1mU/10 μl) in the presence of 0.1% SDS and 0.1M sodium citrate pH 5.5, at37° C. for 12 hours. Equal amounts of protein (3.5 μl) were analyzed on10% SDS-PAGE in accordance with the procedure described in Laemmli etal., Nature (London) 22:680-685 (1970).

PAP and PAP-v encode precursor proteins of 33 and 34 kD, respectively,and both precursors are processed to a 32 kD form after incubation withmembranes. The processed proteins are still larger than the mature form(29 kD), indicating that the PAP precursor undergoes furtherpost-translational processing. PAP does not contain any N-linkedglycosylation sites and the size of the in vitro translated proteins didnot change after treatment with Endo H, which removes carbohydrate.These results indicated that the PAP precursors contain an N-terminalsignal sequence which is co-translationally processed, and anothersequence, which is post-translationally removed. Further evidence forC-terminal processing was obtained from X-ray structure analysis, whichshowed that mature PAP is 29 amino acids shorter at its C-terminus thanthe sequence predicted from the cDNA. See Monzingo et al., J. Mol. Biol.233:705-715 (1993).

C. Growth of transformed yeast: In the presence of 2% raffinose, anon-repressing, non-inducing carbon source relative to GAL geneexpression, the growth of yeast transformants containing NT 123 or NT124was indistinguishable from the transformants containing the vectoralone. Growth of transformed yeast containing NT123 was arrested uponaddition of the inducer, galactose, to the medium. Cells containingNT123 or NT124 did not grow on plates containing galactose. In theliquid medium, however, the extent of inhibition was greater with NT123than NT124, possibly due to lower levels of mature PAP produced in yeastcontaining NT124. PAP expression was detected within 2 h of galactoseaddition to the medium. Maximal levels were reached in 6 to 8 h.Immunoblot analysis using antibodies against PAP, detected a maximal PAPlevel of 1 μg/mg yeast protein in NT123 transformants and 250 ng/mgyeast protein in NT124 transformants. These results were consistent withproduction of active PAP in yeast.

D. Mutagenesis of PAP plasmids. To isolate PAP mutants nontoxic toyeast, the expression plasmids containing PAP (NT123) or PAP-v (NT124)were mutagenized using hydroxylamine, transformed into yeast and cellswere plated on medium containing glucose and replica plated to galactoseconhining plates. About 10 μg of the purified plasmid DNA were added to500 μl of freshly prepared hydroxylamine solution (0.35 ghydroxyiamine-HCl and 0.09 g NaOH in 5 ml of water) and incubated at 37°C. for 20 h. To stop the mutagenesis, 10 μl of SM NaCl, 50 μl of 1 mg/mlBSA and 1 ml of 100% ethanol were added and the mutagenized DNA wasprecipitated by incubation at -70° C. for 10 minutes. The DNA wasresuspended in TE and precipitated again. The DNA was then transformedinto yeast and plated on uracil minus medium containing 2% glucose andreplica plated on medium containing 2% galactose. The colonies that grewon galactose were analyzed for PAP expression by ELISA described inLodge et al., sum., and by immunoblot analysis to identify the mutantswhich expressed hydroxylamine generated mutant PAP.

E. Growth of mutant yeast: Growth of mutants derived from NT123 ongalactose containing medium was indistinguishable from growth onraffinose containing medium. Similar results were obtained with mutantsderived from NT124. Analysis of protein accumulation in yeast indicatedthat the expression of wild type PAP, but not the hydroxylaminegenerated mutant PAP, resulted in decreased protein accumulation inyeast (data not shown).

After mutagenesis, the colonies growing on uracil deficient galactoseplates were analyzed for PAP expression by ELISA using PAP antibodiesand the positives were further analyzed by immunoblot analysis. Of atotal of 28 mutants from NT123 mutagenesis, six different isolatesexpressed proteins which cross-reacted with PAP antibodies. Out of 44mutants isolated from NT124 mutagenesis, 24 different isolates producedproteins which cross-reacted with PAP antibodies. Four mutants(HMNT123-1, 124-6, 124-7, and 124-1) produced proteins which were largerthan the mature form of PAP (29kD), suggesting that the processing ofPAP to the mature form is blocked in these mutants. Two mutants(HMNT123-2 and 123-3) produced proteins that co-migrated with the matureform of PAP, while several others (HMNT123-4, 123-5, 123-6, 124-2 and124-3), produced smaller proteins. The protein expression levels in themutants ranged from 0.005 to 0.08% of total soluble protein.

F. Nucleotide sequence analysis of PAP mutants: The positions of theamino acid alterations in the PAP mutants were identified by sequenceanalysis of the plasmids rescued from yeast. Plasmids were isolated fromthe mutants, transformed into E. coli according to the procedure setforth in Rose et al., supra., and sequenced using the Sequenase 2.0 DNAsequencing kit (USB). See Robzyk et al., Nucl. Acids Res. 2, 3790(1992). Sequence analysis of HMNT123-2 revealed that it contains asingle point mutation, changing the glutamic acid at position 176 tovaline(E176V) at the putative active site (Table II). HMNT123-2 produceda protein of the same size as the wild type PAP. Glutamic acid atposition 176 (E176) is highly conserved among all RIPs sequenced to dateand it is proposed to be at the active site cleft of PAP (4). SeeStevens et al., Experientia 3:257-9 (1981). HMNT123-6, HMNT124-2 andHMNT124-3 all had a point mutation near the C-terminus which introduceda stop codon instead of a tryptophan at position 237 (W237) (Table II).As a result of this mutation, 26 amino acids were deleted from theC-terminus of the mutant PAP, and a truncated protein was produced.HMNT123-5 contained a frameshift mutation, which deleted two nucleotides(GA) at about the codon for Glul84 (GAG), whereby the reading frame wasaltered and the Asn190 codon became TAA, because the reading frameshifted to the -1 position, resulting in expression of a truncatedprotein. A point mutation in HMNT124-1 changed the glutamic acid atposition 97 to lysine (E97K) (Table I). HMNT123-1 also contained asingle point mutation, at position 75, changing glycine to valine(G75V). Both of these mutants expressed a larger protein than purifiedmature PAP, suggesting that processing of PAP is inhibited in thesemutants.

To confirm that the observed mutant phenotypes were due to the mutationsidentified in the PAP sequence, and not due to a chromosomal mutation,each mutant PAP plasmid was isolated and retransformed into the hoststrain, W303, and URA+ transformants were selected. These transformantsgrew at wild type rates on galactose containing medium, indicating thatthe ability of the transformants to survive induction of PAP expressionis plasmid-linked.

                  TABLE II                                                        ______________________________________                                        Mutations which abolish the toxicity of PAP to eukaryotic cells               HMNT123-1       Gly-75 (GGT) → Val (GTT)                                                HMNTI23-2 Glu-176 (GAG) → Val (GTG)                     HMNT123-4 Trp-208 (TGG) → Stop (TAG)                                   HMNT123-5 Glu-184 (GAG) → Glu (GAA)                                    HMNT123-6 Trp-237 (TGG) → Stop (TAG)                                   HMNT124-1 Glu-97 (GAA) → Lys (AAA)                                     HMNT124-2 Trp-237 (TGG) → Stop (TAG)                                   HMNT124-3 Trp-237 (TGG) → Stop (TAG)                                   HMNT124-13 Leu-202 (CTT) → Phe (TTT)                                 ______________________________________                                    

G. Enzymatic activity of PAP mutants: An in vitro translation assay wasused to compare the enzymatic activity of PAP mutants. Brome mosaicvirus (BMV) RNA was translated in the rabbit reticulocyte lysate system(Promega) in the presence of extracts from yeast containing differentamounts of PAP, as described in Lodge et al., supra. PAP levels in yeastwere quantitated by ELISA (Lodge et al., supra.). The inhibition curveswere linear in the range of 0. 1 to 1 ng PAP/ml. Table III shows theresults of the protein synthesis inhibition assay carried out in thepresence of 0.2 ng/ml PAP from yeast. The amount of total protein andPAP were adjusted to 87 ng/ml and 0.2 ng/ml, respectively in eachextract by adding either wild type yeast extract or RIPA buffer. Inprevious experiments, when in v translation was performed in thepresence of 0.2 ng/ml BSA, no inhibition of translation was observed.When 0.2 ng/ml protein from nontransformed yeast (WT) were added, aslight inhibition of translation was observed. Translation was inhibitedin the presence of 0.2 ng/ml of: (1) purified PAP added to wild typeyeast extract (WT+PAP); (2) protein extracts from yeast containing NT123or NT124; and (3) protein extracts from yeast containing thehydoxylamine generated mutants HMNT123-3, HMNT124-1, HMNT124-3 andHMNT124-13. In contrast, protein extracted from HMNT123-2 did notinhibit protein synthesis in the reticulocyte lysate system. Similarresults were obtained when in vitro translation experiments wereperformed using 0.1 ng/ml PAP.

                  TABLE III                                                       ______________________________________                                        Inhibition of protein synthesis by PAP mutants                                                         Protein synthesis (cpm                                 Protein added to translation medium incorporated)                           ______________________________________                                        No RNA                2,246 ± 204                                            BSA 244,956                                                                   WT 176,723 ± 713                                                           PAP + WT 146,660 ± 2474                                                    NT123 110,007 ± 445                                                        HMNT123-2 213,952 ± 767                                                    HMNT123-3 134,202 ± 5522                                                   HMNT124  84,959 ± 661                                                      HMNT124-1 119,529 ± 2094                                                   HMNTI24-3 132,955 ± 3739                                                   HMNT124-13 145,899 ± 4457                                                ______________________________________                                    

EXAMPLE 2 Expression of PAP Min Transgenic Tobacco

Mutant PAPs were engineered for constitutive expression in plants todetermine if they would be non-toxic to plants, and if they could retainPAP antiviral properties.

In order to insert the PAP genes into the plant expression vectors, theplasmid DNA encoding the mutant PAPs was isolated from yeast,transformed into E. coli as described in the Example 1. The plasmid DNAencoding the mutant PAPs was isolated from E. coli digested withHindIII, the HindIII site was filled in with klenow DNA polymerase andthe plasmid was digested with SacI to isolate the 772 bp SacI/HindIIIfragment encoding the mutant PAP. The SacI/HindIII fragments encodingthe mutant PAPs were cloned into pMON8443 (Lodge et al., Proc. Natl.Acad. Sci. USA 9:7089-7093 (1993)) after digestion with SacI and SmaI toremove the wild type PAP cDNA inserts. The SacI/HindIII fragment fromHMNT123-2 was cloned into Sacl/Smal digested pMON8443 to generate NT144and the Sacl/HindlU fragment from HMNT124-3 was cloned into SacI/SmaIdigested pMON8443 to generate NT145. NT146 and NT147 were generated byreplacing the 772 bp SacI/HindIII fragment of the wild type PAP cDNAinsert in pMON 8443 with the 772 bp SacI/HindIII fragment of HMNT123-2and HMNT124-3, respectively.

Plasmids NT144, NT145, NT146 and NT147 were mobilized into the ABIstrain of Agrobacterium tumefaciens for transformation into tobacco andpotato (Lodge et al., 1993). For transformation of N. tabacum, youngleaves from one month old tobacco plants were covered with water for20-30 min. The water was drained off and the leaves were covered with10% chlorox and 0.04% Tween 20 for 15 minutes and then rinsed threetimes with water. Leaf disks were cut with an autoclaved single holepunch and placed upside down on MS104 medium (4.4g/l MS salts, 30 g/lsucrose, B5 vitamins, 0.1 mg/l NAA and 1.0 mb/l BA) for 1 day forpreculture. The precultured disks were placed in a 50 ml tube andinoculated with an overnight culture of Agrobacterium that was diluted1:5. The tube was inverted several times. The disks were then taken outand blotted on sterile filter paper and placed upside down on MS104feeder plates with a filter disk to co-culture for 2 days. Leaf diskswere then transferred to MS104 media with selection (100 μg/ml kanamycinand 300 μg/ml cefataxime) and placed in an incubator. Calli and shootsappeared in about three weeks. Shoots were transferred to plantcons withMSO media (4.4 g/l MS salts, 30 g/l surose and B5 vitamins) withselection. Roots formed in two weeks, rooted shoots were thentransferred to soil and kept in a high humidity environment. Theregenerated plants were then screened by ELISA for the presence ofneomycin phosphotransferase (NPTII) to identify the expressors.

Transformation frequencies of N. tabacum cv Samsun typically rangebetween 10 to 12% (number of transgenic plants obtained per leaf disk).As previously reported the transformation frequency of N. tabacum wassignificantly reduced when vectors containing the wild type PAP(pMON8443) or the variant PAP (pMON8442) were used in transformation(Lodge et al., 1993). In contrast, as shown below in Table IV, nodecrease in transformation frequency was observed when vectorscontaining the nontoxic mutant PAPs were used in the transformation.

                  TABLE IV                                                        ______________________________________                                        Plasmid    Frequency of transformation                                        ______________________________________                                        NT144      13%                                                                  NT145 11%                                                                     NT147 12%                                                                   ______________________________________                                    

As previously reported, the transgenic plants expressing wild type PAPor the variant PAP showed growth reduction, chlorosis and mottling ontheir leaves (Lodge et al., 1993). In contrast, the transgenic plantsexpressing the mutant PAPs were phenotypically normal. They grew at thesame rate as the wild type plants and showed no chlorosis or mottling ontheir leaves, indicating that the expression of the mutant PAPs is nottoxic to transgenic plants. The mutant PAPs were also expressed in E.coli and their expression did not affect the growth rate of E. colicells, indicating that they are not toxic to E. coli.

EXAMPLE 3 Antiviral Activity of Mutant PAP Expressed in TransgenicTobacco

Transgenic tobacco (N. tabacum cv Samsun) plants were assayed by ELISA(Lodge et al., 1993) to determine the level of expression of the mutantPAPs. In Table V, the level of expression of the mutant PAP is comparedwith the level of expression of the variant PAP (pMON8442) (Lodge etal., 1993) expressed in transgenic plants.

                  TABLE V                                                         ______________________________________                                        Level of PAP expression in transgenic tobacco                                       Plant number      Level of expression                                   ______________________________________                                        NT144-12            1.5 μg/mg                                                NT144-13 0.9 μg/mg                                                         NT145-13 4.4 ng/mg                                                            pMON8442(26139-11) 9.6 ng/mg                                                ______________________________________                                    

As shown in Table V, the transgenic plant containing the C-terminaldeletion mutant (NT145-13) expressed similar levels of mutant PAP as theplant expressing the PAP variant (pMON8442) (Lodge et al., 1993). Incontrast, transgenic plants containing the active site mutant (NT144)expressed significantly higher levels of the mutant PAP.

To test if the PAP mutants (the NT144 and NT145 constructs) hadantiviral activity in vitro, wild type tobacco plants were inoculatedwith potato virus X (PVX) in the presence of protein extracts fromplants expressing the mutant PAP, the PAP-v (pMON8442) andnontransformed (wild type) tobacco. PAP levels in the transgenic plantswere quantitated by ELISA. The level of PAP expression in line 145-13was 4.4 ng/mg and the level of PAP expression in line 144-12 was 1.5pg/mg. Plants were inoculated with extracts from transgenic plantscontaining 5 ng PAP per leaf and 1.1 mg total protein. Protein extractwas prepared from nontransformed tobacco leaves (WT). Fifty μl of 1μg/ml PVX was inoculated onto tobacco leaves in the presence ofdifferent amounts of total protein from nontransformed tobacco, rangingfrom 6.7 μg to 1.1 mg. Twenty tobacco plants were inoculated with 50 μlof 1 μg/ml PVX in the presence of 6.7 μg-1.1 mg of total protein fromnontransformed plants. As shown in Table VI, all WT plants becameinfected with PVX and showed local lesions, systemic symptoms and virusaccumulation in the leaves above the inoculated leaves (systemicleaves). These results demonstrate that protein extracts fromnontransformed tobacco plants do not have any effect on PVX infection.When protein extract from nontransformed tobacco plants was used in thepresence of 5 and 10 ng purified PAP (WT+PAP), lower numbers of PVXlesions were observed on inoculated leaves, indicating that tobaccoplants were protected from PVX infection in the presence of purifiedPAP. However, although fewer lesions were obtained on the inoculatedleaves of these plants, they showed systemic symptoms and similar levelsof PVX antigen as the plants inoculated with PVX in the presence ofextracts from nontransformed tobacco plants (WT).

                  TABLE VI                                                        ______________________________________                                        Effects of PAP mutants on PVX infection of tobacco leaves.sup.1.                                                  PVX antigen                                 Plant Extract.sup.a PAP ng/(leaf) mean no. of lesions.sup.b level                                               (ng/mg).sup.c                             ______________________________________                                        WT.sup.4  0          66.6 ± 10.1                                                                             4.4 ± 1.4                                  WT + PAP.sup.e 5 9.0 ± 2.0 3.1 ± 1.9                                     10 1.5 ± 2.0 2.8 ± 2.6                                                 26139 5 1.8 ± 2.9 NA                                                       145-13 5 12.5 ± 7.4  0.2 ± 0.3                                          144-12 5 57.8 ± 7.4  3.2 ± 2.5                                           10 56.1 ± 4.9  2.5 ± 1.4                                                20 55.5 ± 13.8 4.3 ± 1.4                                                50 53.3 ± 14.9 2.8 ± 0.3                                                100 68.0 ± 11.7 3.0 ± 0.9                                            ______________________________________                                         .sup.a Plant extract was prepared from either nontransformed or               transformed tobacco leaves with plant expression vector (pMON8442, NT145,     and NT144).                                                                   .sup.b The number of lesions were counted at 9 days postinoculation.          .sup.c Three leaf discs in a tube were taken from 1st, 2nd and 3rd            systemic leaves at 12 days post inoculation and then homogenized in ELISA     buffer. The average levels of PVX antigen were quantified by ELISA. The       amount of total proteins in each extract were quantified by BCA reagent       (Pierce).                                                                     .sup.d Protein extract was made from nontransformed tobacco leaves.           .sup.e PAP (Calbiochem) was added to a protein extract from nontransforme     tobacco leaves.                                                               .sup.1 Twenty plants for wildtype(wt), ten plants for wt + PAP and five       plants for each transgenic protein extract were used. Two leaves from eac     plant were inoculated with 50 μl of PVX (1 μg/ml) in the presence o     different amount of PAP or PAP mutants.                                  

When PVX was inoculated in the presence of 5 ng protein from thetransgenic plant (26139) expressing the variant PAP (pMON8442),significantly lower numbers of lesions were observed on the inoculatedleaves and these plants escaped systemic infection. Similarly, when PVXwas inoculated in the presence of 5 ng protein from transgenic plant(145-13) expressing the C-terminal deletion mutant, significantly fewerlesions were obtained. These plants did not show systemic symptoms andthe PVX antigen levels were significantly reduced on the inoculatedleaves. In contrast, when PVX was inoculated in the presence of 5 to 100ng protein from transgenic plant expressing the active site mutant(144-12), the numbers of lesions observed on the inoculated leaves wassimilar to the numbers of lesions observed on plants inoculated in thepresence of protein from nontransformed tobacco plants (WT). Systemicsymptoms were observed on these plants and PVX antigen levels in thesystemic leaves were comparable to the antigen levels in plantsinoculated with PVX in the presence of extracts from nontransformedtobacco plants. These results demonstrate that the C-terminal deletionmutant which is enzymatically active in vitro retains its antiviralactivity in vitro. In contrast, the active site mutant which isenzymatically inactive in vitro, does not retain its antiviral activityin vitro, suggesting that the enzymatic activity of PAP is critical forantiviral activity in vitro.

EXAMPLE 4 Expression of PAP Mutants in Transgenic Potato

Potato stems were cut into 3 mm pieces and placed in sterile water.Agrobacterium containing NT144, Nt145, NT146 and NT147 was grownovernight. Cells were spun down and resuspended in 10 ml of water.Agrobacterium was diluted again 1:10 in water. Water was removed frompotato stem explants and the diluted Agrobacterium was added. The stemexplants were incubated with Agrobacterium for 15 min. The bacteria wereremoved and the explants were placed on 1/10 MSO plates that had beencovered with sterile Whatman #1 filters. MSO contains 4.4 g MS salts, 30g sucrose and 1 ml B5 vitamin (500×) in a 1 liter volume, pH 5.7. Aftera two day co-culture period in the dark, the explants were placed on PCmedia, containing MSO plus 0.5 mg/l zeatin riboside (ZR), 5 mg/l AgNO₃and 0.1 mg/l NAA (naphthaleneacetic acid) 100 mg kanamycin and 300 mgcefataxirm per liter, for four weeks. After 4 weeks, the explants wereplaced on PS media which contains MSO plus 5 mg/l ZR, 0.3 mg/lgiberellic acid, 100 mg kanamycin and 300 mg cefataxime per liter.Shoots began to appear in four to eight weeks. Shoots were then removedand placed in plantcons containing PM media (4.4 g MS salts, 30 gsucrose, 0.17 g NaH₂ PO₄ H₂ O, 1 ml thiamine HCl and 0.1 g inositol in a1 liter volume, pH 6.0 and 0.2% Gehrite agar). Plants were then placedin soil, hardened off and analyzed by NPTII ELISA to identify thetransgenic plants. Transgenic potato plants were then analyzed by ELISAfor PAP expression. Transgenic potato plants expressing NT144, NT145 andNT 146 were identified by ELISA. The transformation frequencies were notaffected when constructs containing mutant PAPs were used and thetransgenic plants expressing mutant PAPs were phenotypically normal,indicating that the expression of the mutant PAPs is not toxic topotato.

EXAMPLE 5 Expression of PAP Mutants in Transgenic Turfgrass

Mutant PAPs were engineered for constitutive expression in monocots.Creeping bentgrass (Agrostis palustris, Huds.), which is a turfgrassused in golf courses, fairways, tees and lawns, was used as the monocotspecies for transformation. In order to construct an expression vectorfor monocots, NT168 was created by cloning the promoter and the firstintron of the maize ubiquitin gene (Toki et al., Plant Physiol.100:1503-1507, 1992) into pMON969. pMON969 was digested with HindIII andBglII to remove the CaMV 35S promoter region. The plasmid pAHC20,containing the ubiquitin promoter and the first intron (Toke et al,1992) was digested with HindIII and BamHI to isolate the 2016 bpHindIII/BamHI fragment which was ligated to HindIII/BglII fragment ofpMON969 to generate NT168. The cDNA fragments encoding the mutant PAPswere isolated by digesting NT144 and NT145 with BgIII and BamHI andcloned into the BamHI site of NT168. The monocot expression vectorscontaining the mutant PAP cDNAs were then used in transformation alongwith pSLI2011, which contains the selectable marker, the bar gene(Hartmann et al., 1994 Biotechnology 12:919-923. Turfgrasstransformation was carried out using two different methods, biolistictransformation using the particle gun and by protoplast transformationas described below.

Embryogenic callus cultures were initiated from surface sterilized seedsof 7 creeping bentgrass cultivars: `Cobra`, `Emerald`, `PennLinks`,`Providence`, `Putter`, `Southshore`, and `SR1020` and used in biolistictransformation, as described in Hartmann et al., Biotechnology12:919-923 (1994). Callus initiation media were MS basal medium and MSvitamins, supplemented with 100 mg L⁻¹ myo-inositol, 3% sucrose, andeither 150 mg l⁻¹ asparagine and 2 mg L⁻¹ 2,4-D for MSA2D, or 500 mg L⁻¹casein hydrolysate, 6.6 mg L⁻¹ dicamba, and 0.5 mg L¹ 6-BA for MMS.Media were solidified with 0.2% Phytagel® (Sigma). After 4 to 6 weeks inthe dark at 25° C., embryogenic callus lines were selected andtransferred to fresh medium. Suspensions were established fromembryogenic callus cultures by adding 1 to 2 g callus to 250 ml flaskswith 50 ml liquid media, incubate in the dark at 25° C. with shaking at120 rpm and subcultured twice a week.

Plates were prepared for particle bombardment by placing 1 ml ofsuspension cells on 5.5 cm filter disks in plates containing MSA2D mediawith the addition of 0.4 M mannitol. Plates were prepared 20 h prior tobombardment and kept in the dark. Gold particles were prepared byheating at 95° C. in 100% ethanol for 30 min, centrifuged briefly andresuspended in fresh ethanol. The particles were sonicated for 10-30 minin a water bath, washed 3 times in sterile, distilled water, andresuspended in water. DNA samples consisting of 50 μl (5 mg) goldsuspensions, 10 μg target DNA, 50 μl 2.5M CaCl₂, and 20 μl 0.1 Mspermidine, were vortexed, centrifuged, and resuspended in ethanol. Theethanol wash was repeated for a total of 3 times. The final pellet wasresuspended in 30 μl ethanol, and 5 μl of DNA solution were used pershot. Bombardment was carried out using the Bio-Rad PDS-1000, HeBiolistic Delivery System at 1100 psi. Calli from the bombardmentexperiments were plated out on MSA2D medium containing 2 or 4 mg/l ofbialaphos for selection 3-4 days after bombardment and continued for 8weeks without transfer. After 8 weeks on plate selection, calli weretransferred to MS media without hormones for regeneration. Regeneratesappeared within 2-8 weeks. Shoots were transferred to Plantcons®containing MS medium and roots appeared within 2-4 weeks.

For protoplast transformation, protoplast isolation was performed fourdays after subculture. Cells were incubated with filter-sterilizedenzyme solution containing 1% (w/v) Cellulase Onozuka RA (YakultPharmaceutical Co. LTD), 0.1% Pectolyase Y-23 (Seishin PharmaceuticalCo. LTD), and 0.1% MES (2-[N-morpholino]ethane-sulfonic acid) (Sigma) inculture media (MSA2D or MMS with 5% mannitol) for 4 hours at 28° C. withshaking at 50 rpm. About 1 g fresh weight of suspension cultures wastreated with 10 ml of enzyme solution. Protoplasts were filtered throughMiracloth and washed twice with culture medium containing 5% mannitol.Mannitol was used as an osmotic stabilizing agent. Protoplasts werecultured using a feeder layer system (Rhodes et al., 1988). The washed,filtered protoplasts were pipetted onto a black nitrocellulose membrane(Lee et al., 1989) placed over a feeder layer of suspension cells whichhad been spread on 5% mannitol culture medium. One week later, themembranes with protoplasts were transferred to a fresh feeder layer on3% mannitol culture plates. Protoplasts were removed from the feederlayer 2 weeks after isolation. Plating efficiency was determined bydividing the number of visible colonies 3 weeks after isolation by thetotal number of protoplasts plated. Plants were regenerated by placingprotoplast derived calli on MS medium without hormone or with 1 mg L⁻¹6BA orkinetin. After 4 to 5 weeks shoots were transferred to Plantcon®with MS medium containing no hormone for rooting. Protoplasts weretransformed using either PEG following the protocol of Negrutiu et al.,(1987), or electroporation at 170 volts cm⁻¹ using a Gene-Pulster(Bio-Rad). In PEG experiments, freshly isolated protoplasts wereresuspended at a density of 1×10⁷ protoplasts per ml in 5% mannitolcontaining 15 mM MgCl₂ and 0.1% MES. Approximately 0.3 ml protoplastswere incubated with 20 to 40 μg plasmid DNA and 13% PEG for 10 to 15min., diluted stepwise and resuspended in culture medium with 5%mannitol (pH 5.8) after centrifugation. In electroporation experiments,protoplasts were resuspended at a density of 5×10⁶ protoplasts per ml incold filter sterilized electroporation buffer containing 5.2 g L⁻¹ KCl,0.835 g L⁻¹ CaCl₂, 0.976 g L⁻¹ MES and 5% mannitol at pH 5.8. About 0.8ml protoplasts were mixed with 20 μg DNA by inversion, electroporated at170 volts cm⁻¹ and placed on ice for 15 min., then diluted to a total of3 ml with culture medium containing 5% mannitol. Selection with 4 mg L⁻¹of bialaphos was initiated 16 days after protoplast isolation andtransformation. Resistant colonies were selected on MS medium withouthormone, with 6-BA or kinetin as described above. Shoots weretransferred to Plantcons® for rooting. A commercial formulation ofbialaphos under the trade name Herbiace® (Meiji Seika Kaishya, LTD.) wasused in greenhouse herbicide tests. Herbicide rates for Herbiace® wereestablished using control plants, and were based on the commercial rateof 0.75 lb AI/acre (1× the field rate). The herbicide was applied to allthe tillers above ground with an artist's paint brush at the rate of 120ml per flat. Dimension of the flat is 0.1431 m² and it holds 96 or 24plants.

EXAMPLE 6

Expression of Pap Mutants in Transgenic Tobacco Plants and Resistance toViral Infection

A. Expression of PAP mutants in transgenic tobacco

To determine if enzymatic activity of PAP is required for its antiviralactivity, the cDNA encoding the active-site mutant NT123-2 was clonedinto the plant expression vector pMON8443 after removing the wild typePAP insert, to generate NT144, as described in Example 2. Similarly, thecDNA encoding the C-terminal deletion mutant, NT124-3 was cloned intopMON8443 to generate NT145 and NT147, as described in Example 2.Expression of the mutant PAPs was driven by the enhanced CaMV35Spromoter. NT144, NT145 and NT147 were mobilized into Agrobacteriumtumefaciens for transformation into tobacco. Transformation frequenciesof Nicotiana tabacum cv Samsun typically range between 10 to 12% basedon the number of transgenic plants obtained per leaf disk (Lodge et al.,1993). The transformation frequency was 13% using NT144 and 11% usingNT145. The transgenic plants expressing the active-site mutant or theC-terminal deletion mutant were phenotypically normal. They grew at thesame rate as wild type plants and did not show chlorosis or mottling intheir leaves, indicating that the expression of the mutant PAPs was nottoxic to transgenic tobacco. These results are in contrast to thepreviously reported results (Lodge et al., 1993) in which thetransformation frequencies of N. tabacum were reduced to 0.7% when usinga vector containing PAP (pMON8443), and to 3.7% when using a vectorcontaining PAP-v (pMON8442), both of which are enzymatically active(Lodge et al., 1993). Lodge et al. did not recover any transgenic plantsexpressing high levels of PAP, and the transgenic plants expressing highlevels of PAP-v showed growth reduction, chlorosis, and mottling intheir leaves.

Regenerated transgenic plants were first analyzed for expression ofneomycin phosphotransferase (NPTII) by ELISA. The NPTII positive plantswere then analyzed for PAP expression by ELISA and immunoblot analysis.Eleven different transgenic plants expressed detectable levels of theactive-site mutant by ELISA. Plants expressing the active-site mutantPAP produced a 29 kDa protein which comigrated with mature PAP,indicating that the active-site mutant PAP is fully processed to themature form in transgenic plants (data not shown). Transgenic plantsexpressed significantly higher levels of the active-site mutant than theplants expressing PAP or PAP-v. No bands corresponding to PAP weredetected in wild type tobacco or in transgenic tobacco expressingβ-glucuronidase. The C-terminal deletion mutant was expressed atsignificantly lower levels than the active-site mutant.

B. Antiviral activity of the active-site mutant in transgenic tobacco

In order to determine if transgenic lines expressing the active-sitemutant PAP are resistant to virus infection, progeny of transformedplant lines were inoculated with PVX. Self-fertilized progeny werescreened for the presence of PAP by ELISA. PAP levels in the progeny ofthe transgenic lines varied depending on the age of plants and growthconditions. The degree of variability in PAP levels was similar to thatpreviously reported for transgenic lines expressing PAP or PAP-v (Lodgeet al., 1993). Ten progeny-from each transgenic line expressing theactive-site mutant PAP (144-1 and 144-7), PAP-v (26139-19), PAP(33617-11) and 10 nontransformed tobacco plants were inoculated with 1μg/ml PVX. Symptom development on both inoculated and systemic leaveswas monitored visually each day up to 21 days post-inoculation. Inaddition, disks from the inoculated and from the first, second and thirdsystemic leaves of each plant were sampled at 12 days post-inoculationin order to quantitate virus replication and spread.

As shown in Table VII, transgenic plants expressing PAP-v or the wildtype PAP did not develop any lesions on the inoculated leaves at ninedays post-inoculation. In contrast, transgenic plants expressing theactive-site mutant had as many lesions on their inoculated leaves as thecontrol plants. ELISA analysis of systemic leaves showed that 90% ofwild type tobacco plants were systemically infected by PVX at 12 dayspost-inoculation, while only 30 and 40% of the transgenic plantsexpressing PAP and PAP-v, respectively, showed systemic PVX infection.In contrast, 100% of the plants expressing the active-site mutant wereinfected with PVX (Table VI). Similar results were obtained when plantswere scored again at 21 days post inoculation.

                  TABLE VII                                                       ______________________________________                                               PAP      Level of PAP                                                                             Number % of plants showing                           Plant Line expressed (ng/mg).sup.a of lesions.sup.b systemic infection.s                                      up.c                                        ______________________________________                                        WT              0          77 ± 12                                                                            90                                           26139-19 PAP-v  5.6 ± 2.6 0 **    30 **                                    33617-11 PAP  0.6 ± 0.02 0 **    40 *                                      144-1 E176V 43.8 ± 4.8 78 ± 16 100                                      144-7 E176V 46.2 ± 5.6 72 ± 11 100                                    ______________________________________                                         .sup.a PAP levels were quantitated by ELISA after taking four leaf disks      from twenty plants per line. Mean values ± SD are shown.                   .sup.b Ten plants from each line were inoculated with 50 μl of 1           μg/ml PVX on two leaves per plant. The number of lesions were counted      days post inoculation. Mean values ± SD are shown.                         .sup.c Three leaf disks were taken from 1st, 2nd and 3rd systemically         infected leaves at 12 days post inoculation and viral antigen levels were     quantitated by ELISA. The amount of total protein in each extract was         quantitated using the BCA kit (Pierce).                                       ** Significantly different from wild type at 1% level                         * Significantly different from wild type at 5% level                     

To determine if transgenic plants expressing higher levels of theactive-site mutant are also susceptible to PVX infection, homozygousprogeny (R2 generation) of transgenic link 144-12, which expressed thehighest levels of the active-site mutant PAP were inoculated with 0.5ig/ml PVX. As shown in Table VIII below, transgenic lines producing highlevels of the active-site mutant had the same numbers of lesions as thewild type tobacco plants in their inoculated leaves, while progeny oftransgenic plants which expressed PAP-v or PAP had significantly lowernumbers of lesions. ELISA analysis of the systemic leaves demonstratedthat by 21 days post inoculation, 90% of wild type tobacco plants and100% of the transgenic plants expressing the active site mutant wereinfected with PVX. In contrast, plants expressing PAP or PAP-v had fewerlesions on the inoculated leaves and lower percentages of these plantsbecame systemically infected with PVX.

In additional experiments, progeny of seven different transgenic linesexpressing the active-site mutant were analyzed for their susceptibilityto PVX infection; none of these lines showed resistance to PVX (data notshown).

                  TABLE VIII                                                      ______________________________________                                        Susceptibility of transgenic tobacco plants expressing the                      C-terminal deletion mutant (W237Stop) to PVX infection                                                           % of plants                                    showing systemic                                                           PAP Level of PAP Number infection.sup.c                                    Plant Line                                                                            expressed                                                                              (ng/mg).sup.a                                                                            of lesions.sup.b                                                                     12 dpi                                                                              21 dpi                               ______________________________________                                        WT               0          24 ± 15                                                                            90    90                                    26139-19 PAP-v 9.6  1 ± 2**  10**  30**                                    33617-11 PAP 1.6 11 ± 4**  10*  40*                                        144-12 E176V 1500 23 ± 13 100 100                                          147-19 W237Stop 4.5 12 ± 10**  20**  60                                    145-13 W237Stop 4.4  6 ± 4**  30**  60                                   ______________________________________                                         .sup.a PAP levels were quantitated by ELISA in the primary transgenic         plants.                                                                       .sup.b Eight to ten plants from the homozygous progeny (R2 generation) of     each transgenic line were inoculated with 50 μl of 0.5 μg/ml PVX on     two leaves per plant. The number of lesions were counted 12 days post         inoculation. Mean values ± SD are shown.                                   .sup.c Two leaf disks were taken from first and second systemically           infected leaf from each plant at 12 days postinoculation and two leaf         disks were taken from third and fourth systemically infected leaf at 21       days postinoculation. Viral antigen levels were quantitated by ELISA. The     amount of total protein in each extract was quantitated using the BCA kit     (Pierce).                                                                     **Significantly different from wild type at 1% level                          *Significantly different from wild type at 5% level                      

C. Antiviral activity of C-terminal deletion mutant in transgenictobacco

In order to determine if transgenic lines expressing the C-terminaldeletion mutant are resistant to virus infection, homozygous progeny (R2generation) from transgenic lines 145-13 and 147-19 expressing theC-terminal deletion mutant (W237Stop) were inoculated with 0.5 μg/ml PVXand the numbers of lesions were counted at 12 days post-inoculation. Asshown in Table VIII above, plants from transgenic lines 145-13 and147-19 had significantly lower numbers of lesions on their inoculatedleaves compared to the wild-type plants. At 12 days post inoculation,only 20 and 30% of the plants from the transgenic lines 147-19 and145-13, respectively, showed systemic symptoms and contained PVX antigenby ELISA, while 90% of the control plants were infected with PVX. By 21days post-inoculation, there was an increase in the percentage of plantsfrom lines 147-19 And 145-13 that showed systemic symptoms. As observedin previous tests, progeny of transgenic lines expressing PAP-v and PAPwere protected from PVX infection. Infected plants expressing theC-terminal deletion mutant (W237Stop), PAP or PAP-v showed mildersymptoms compared to the infected wild-type plants or transgenic plantsexpressing the active-site mutant (E176V). ELISA analysis was used toquantitate viral antigen levels in transgenic plants and wild-typeplants at 21 days post-inoculation. PVX antigen levels were lower inplants from lines 147-19, 145-13, and 33617-11 compared to the antigenlevels wild type plants. The percentages of infected plants did notchange when they were scored again at 4 weeks post inoculation.

In additional experiments, a total of six different transgenic linesexpressing the C-terminal deletion mutant were analyzed for theirsusceptibility to PVX infection and four of these lines showedresistance to PVX infection (data not shown).

EXAMPLE 7

Analysis of Fungal Resistance in Transgenic Plants Expressing PAP andPAP Mutants

Seedlings of transgenic tobacco lines expressing PAP, PAP mutants andwild-type tobacco seedlings were used. Four weeks after germinationseedlings were transferred into growth chamber and were grown in thesterile soil at 25° C., 80% relative humidity, and 16-hour photoperiod.Recombinant constructs with chimeric PAP genes were introduced intoAgrobacterium tumefaciens via triparental mating. Agrobacteriumcontaining the modified PAP genes were used to transform Nicotianatabacum cv. Samsun. Kanamycin resistant R₂ transgenic plants wereself-pollinated, and R₃ seedlings were used in the experiments.Transgenic plants from lines 33617 (expressing wild type PAP), NT144(expressing active-site mutant PAP), NT145, and NT147 (both expressingC-terminal deletion mutant PAP) were used.

Four week-old transgenic and control seedlings were transplanted intosterile soil and inoculated with soil-borne fungal pathogen Rhizoctoniasolani. Development of disease symptoms was observed for two weeks andthe seedling mortality rates were calculated. Plants that survived thefungal infection were transplanted into individual pots and samples oftissue were taken for further analysis.

Following inoculation with R. solani, control tobacco seedlings werevery quickly overcome by fungal pathogen. The disease progressedrapidly, affecting more than 30% of control seedlings in six dayspost-inoculation. In contrast, the transgenic lines' susceptibility toinfection was significantly lower. Six days post-inoculation, only 9.5%of the seedlings from the lines with wild-type PAP, about 20% ofseedlings from the C-terminal truncated PAP line, and 23% of theseedlings from the active-site mutant line were affected. The number ofseedlings that survived at different time points is shown in Table IXbelow. All transgenic lines exhibited a delay in appearance of diseasesymptoms and a lower mortality rate.

                  TABLE IX                                                        ______________________________________                                        Progression of disease in transgenic tobacco PAP lines                          infected with Rhizoctonia solani.                                                    Number of seedlings survived post-inoculation                        Tobacco line                                                                           0 days (%)                                                                             6 days (%)                                                                              10 days (%)                                                                           14 days (%)                               ______________________________________                                        wild type                                                                              40 (100) 27 (67.5) 25 (62.5)                                                                             25 (62.5)                                   33617-11 42 (100) 38 (90.5) 35 (83.3) 34 (81.0)                               145-15-3 37 (100) 29 (78.4) 26 (70.3) 23 (62.2)                               147-19-25 39 (100) 32 (82.1) 29 (74.4) 28 (71.8)                              144-12-3 39 (100) 30 (76.9) 29 (74.4) 29 (74.4)                             ______________________________________                                    

In a separate experiment, with a different strain of Rhizoctonia solani,the disease progressed very rapidly, essentially killing the majority ofseedlings in five days. Seedling survival after two weeks of growth inthe infected soil is shown in Table X below. Noticeably, control plants,although not dead at the scoring time point, were extremely stunted, andexhibited very severe disease symptoms. In contrast, seedlings intransgenic lines with truncated PAP showed much less tissue damage.

                  TABLE X                                                         ______________________________________                                        Survival of transgenic tobacco lines with different PAP genes                   in Rhizoctonia solani resistance test                                                               Number of                                                 seedlings survived % seedlings                                              Tobacco line Number of 14 days survived 14 days                               (Samsun) seedlings planted postinoculation postinoculation                  ______________________________________                                        control (n)                                                                           20          2            10                                             control (N) 20 0 0                                                            33617-11 20 8 40                                                              144-12 20 5 25                                                                145-15 20 1 5                                                                 147-19 20 5 25                                                              ______________________________________                                    

Analysis of surviving plants

Analysis of the total cellular protein from transgenic lines wasperformed by separating protein samples on 10% SDS-PAGE using aMini-PROTEAN II electrophoresis cell (Bio-Rad) and proteins weretransferred onto nitrocellulose membrane using Bio-Rad Trans-Blotsemi-dry electrophoretic transfer apparatus according to manufacturer'sinstructions. Western blot analysis was performed using PAP IgG or PR1amonoclonal antibodies. Detection was by enhanced chemiluminescence usingDuPont Renaissance kit.

Western blot analysis of cellular extracts from transgenic plants showedthat the PAP gene is expressed in all plants that survived the fungalinfection. The amount of PAP produced differed among individual plants.In addition, apoplastic fluid was isolated from the same plants andextaacellular proteins were analyzed by staining the native gel withsilver nitrate. Expression of pathogenesis-related proteins (PR) wasdetected in plants expressing pokeweed antiviral protein gene. Westernblot analysis also showed elevated levels of PR1a in surviving plants.

Significant reduction of fungal disease symptoms in transgenic tobaccolines expressing pokeweed antiviral protein was observed. As shown inTables IX and X, transgenic lines with PAP exhibited greater percentageof seedling survival after infection by R. solani. In addition, thedisease progression, represented by the rate of seedling mortality, wasalso slower in transgenic PAP lines. Transgenic line 33617, whichexpressed the wild type PAP, as well as transgenic tobacco lines thatcontained mutant forms of PAP, NT144-12 (which expresses the active sitemutant PAP), NT145-15 and NT147-19 (which expressed a truncated form ofPAP, lacking 25 C-terminal amino acids) showed resistance to fungalinfection.

Expression of the mutant PAP genes in tobacco proved to have absolutelyno detectable phenotypic effect but surprisingly led to the constitutiveexpression of several pathogenesis-related proteins. Some of the genesinduced are known for their anti-fungal activity. In the light of thisobservation, and while not intending to be limited to any particulartheory of operation, Applicant believes that the resistance toRhizoctonia solani infection by tobacco lines expressing mutant PAPgenes of the present invention may be explained by the action of thehost defense genes, and that resistance to fungus infection in plantsexpressing PAP may be conferred by dual action of PAP transgene and anumber of host genes, constitutively expressed in transgenic tobacco.Applicant further believes that the induction of these plant defensegenes further serves to protect transgenic plants against otherpathogens such as bacterial pathogens.

EXAMPLE 8

Isolation of New PAP Mutants by Chromosomal Mutagenesis and Selection inYeast

A. Isolation of PAP mutants

Chromosomal mutagenesis and selection were used to isolate yeast mutantswhich permit cells to grow in the presence of PAP. Constitutiveexpression of PAP in S. cerevisiae is normally lethal. Therefore, thePAP gene was placed under the control of the galactose inducible GALLpromoter. This enables cells carrying the plasmid with the PAP gene togrow normally on glucose when PAP expression is repressed, but killscells grown on galactose when PAP is expressed. We have taken advantageof having an inducible PAP expression system and the toxicity of PAP tonormal yeast cells, to isolate mutants which can grow in the presence ofPAP. Yeast cells carrying a plasmid with the wild-type PAP gene (NT123)were grown to early log phase, pH 7.0, at a density of 1×10⁸ cells/ml.Three, 1 ml, aliquots were removed and used for the mutagenesis.Mutagenesis was performed using either 5 μl or 25 μl of ethylmethanesulfonate (EMS). An unmutagenized aliquot was kept as the controlto examine the frequency of spontaneous mutants. Following the additionof EMS, the cells were incubated at 30° C. for 1 hour, with gentleshaking. The mutagenesis was terminated by the addition of 5% sodiumthiosulfate. The cells were then plated on uracil deficient plates with2% glucose and incubated at 30° C. Based on the number of colonies whicharose on the plates from the mutagenized cells versus the unmutagenizedcontrol, 35% and 98% of the cells were killed with 5 μl and 25 μl ofEMS, respectively. These colonies were replica plated to uracildeficient media with 2% galactose and screened for colonies capable ofgrowing in the presence of PAP. Approximately 13,500 colonies werescreened, and 9 colonies were obtained which were able to grow ongalactose.

The mutants were tested to see if the mutations were chromosomal orplasmid linked. Plasmid segregation was performed on the mutants bygrowing the cells for approximately 50 generations in non-selectivemedia (YEPD), plating them out on YEPD, followed by replica plating thecolonies which, having lost the plasmid, can no longer grow on uracildeficient media. The plasmid segregated cells were transformed withfresh NT 123 plasmid and examined for their ability to grow on uracildeficient media with 2% galactose. Mutants which retained the ability togrow on galactose are chromosomal mutants, while mutants which failed togrow on galactose carry plasmid borne mutations.

The plasmid borne mutants were further characterized by performingimmunoblot analysis on whole cell extracts from the cells expressingthese plasmids. This analysis revealed that 2 of the 7 plasmid mutantswere expressing a truncated form of PAP. The other 5 mutants were notexpressing any PAP protein. The 2 mutants which were expressingtruncated PAP were examined by sequence analysis to determine the sitesof the mutations. One mutant, NT 185, had a point mutation at theC-terminus, changing Lys210 (AAG) to a stop codon (TAG), resulting in adeletion of approximately 3.5 kDa. The other mutant, NT 187 had a changein the N-terminus, changing Try 16 (TAC) to a stop codon (TAA) and thenwas able to restart at Met39, resulting in a 24.8 kDa protein.

B. Construction of E. coli expression vector

To express the N-terminal deleted mature PAP in E. coli cells, NT 187plasmid DNA was digested with BstYI and HindIII restriction enzymes andthe fragment around 830 bp was purified using the Gene Clean kit (Bio101). The purified fragment was ligated to the E. coli expressionvector, pQE31 (QIAGEN Inc.), which was digested with BamHI and HindIIIand then treated with alkaline phosphatase. The resulting plasmid,NT190, contains the N-terminal deletion mutant PAP in the E. coliexpression vector pQE31.

C. Expression of PAP mutants in E. coli.

NT190 was isolated from E. coli DH5a cells and transformed into theexpression host, E. coli M15 (pREP4). M15 cells containing NT190 werecultured on 50 ml of LB medium containing 2% glucose, 100 ;μg/mlampicillin, and 50 μg/ml kanamycin at 37° C. overnight with vigorousshaking. The following day, a large culture (500 ml of LB medium,containing 2% glucose, 100 μg/ml ampicillin, and 50 μg/ml kanamycin) wasinoculated and grown at 37° C. with vigorous shaking until A₆₀₀ reached0.9. IPTG was added to a final concentration of 2 mM, and the culturewas incubated at 37° C. for 5 hours. Cells were harvested bycentrifugation at 4,000× g for 10 min and stored at -70° C.

D. Purification of N-terminal deleted PAP

One gram of E. coli cells was thawed and resuspended in 5 ml of buffer A(6M guanidinium hydrochloride, 0.1M sodium phosphate, and 0.01MTris-HCl, pH 8.0) and stirred for 1 hr at room temperature. E. colilysate was centrifuged at 10,000× g for 15 min at 4° C. and supernatantwas collected. Two ml of a 50% slurry of Ni-agarose resin (QIAGEN Inc.),previously equilibrated in buffer A, were added. After stirring at roomtemperature for 45 min, the resin was carefully loaded into a poly-prepchromatography column (Bio-Rad). The column was washed with 20 columnvolumes of buffer A, and 10 column volumes of buffer B (8M urea, 0.1 Msodium phosphate, and 0.01M Tris-HCl, pH 8.0). Proteins which did notbind the resin were washed with 20 column volumes of buffer C (8M urea,0.1M sodium phosphate, and 0.01M Tri-HCl, pH 6.3). Finally, the boundprotein was eluted with 50 ml of buffer C containing 250 mM imidazoleand analyzed by SDS-PAGE and western blot analysis.

E. Antiviral activity of N-terminal deleted PAP

To determine if N-terminal deleted PAP had anti-viral activity,wild-type tobacco plants were inoculated with 1 μg/ml PVX in thepresence or absence of N-terminal deletion mutant purified from E. col.PAP concentration was determined by ELISA and by SDS-PAGE. 15 ng/td and1.5 ng/μl mutant PAP were applied to tobacco leaves in the presence orabsence of 1 μg/ml PVX. As shown in Table XI, tobacco plants inoculatedwith PVX in the presence of 1.5 or 15 ng/μl N-terminal deleted PAPshowed fewer lesions on their inoculated leaves compared to plantsinoculated with PVX in the absence of mutant PAP. Furthermore, as shownin Table XII, none of the plants inoculated with PVX in the presence of15 ng/μl mutant PAP, and only 13% of plants inoculated with PVX in thepresence of 1.5 ng/μl mutant PAP showed systemic PVX symptoms, while100% of the plants inoculated with PVX in the presence of buffer aloneshowed systemic PVX symptoms. These results indicate that exogenouslyapplied N-terminal deleted PAP protects tobacco against PVX infectionand is thus anti-viral.

                  TABLE XI                                                        ______________________________________                                        Susceptibility of tobacco plants to PVX in the presence of                      exogenously applied N-terminal deleted PAP                                       Protein applied.sup.a                                                                       PVX                                                          (ng/μl) (μg/ml) Mean # of lesions.sup.b                               ______________________________________                                        none           1       20 ± 16                                               PAP (1.5) 1 2 ± 2                                                          PAP (15) 1 2 ± 2                                                         ______________________________________                                         .sup.a Two leaves from each plant were inoculated with 50 μl of PVX (1     μg/ml) in the presence of 1.5 or 15 ng/μl Nterminal deleted PAP.        Twelve plants were inoculated with PVX in the presence of buffer alone        ("none") and 8 plants were inoculated with PVX in the presence of 50 μ     of 1.5 or 15 ng/μl of mutant PAP.                                          .sup.b The number of lesions were counted at 7 days postinoculation. Mean     values ± SD are shown.                                                

                  TABLE XII                                                       ______________________________________                                        Percentage of plants showing systemic symptoms in the presence of              exogenously applied N-terminally deleted PAP                                     Protein applied.sup.a                                                                       PVX     % plants showing systemic                             (ng/μl) (μg/ml) symptoms.sup.b                                        ______________________________________                                        none          1       100                                                       PAP (1.5) 1 13                                                                PAP (15) 1 0                                                                ______________________________________                                         .sup.a Two leaves from each plant were inoculated with 50 μl of PVX (1     μg/ml) in the presence of 1.5 or 15 ng/μl of Nterminal deleted PAP.     Twelve plants were inoculated with PVX in the presence of buffer alone        ("none") and 8 plants were inoculated with PVX in the presence of 50 μ     of 1.5 or 15 ng/μl of mutant PAP.                                          .sup.b Systemic symptoms were scored 11 days post inoculation.           

Applicant's copending patent application Ser. Nos. 08/500,611 and500,694, filed Jul. 11, 1995, and PCT application No. PCT/US96/11546,filed Jul. 11, 1996, are herein incorporated by reference in theirentireties.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All these publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Various modifications of the invention described herein will becomeapparent to those skilled in the art. Such modifications are intended tofall within the scope of the appending claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1379 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 225..1163                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 225..290                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTATGAAGTC GGGTCAAAGC ATATACAGGC TATGCATTGT TAGAAACATT GA -            #TGCCTCTG     60                                                                 - - ATCCCGATAA ACAATACAAA TTAGACAATA AGATGACATA CAAGTACCTA AA -            #CTGTGTAT    120                                                                 - - GGGGGAGTGA AACCTCAGCT GCTAAAAAAA CGTTGTAAGA AAAAAAGAAA GT -            #TGTGAGTT    180                                                                 - - AACTACAGGG CGAAAGTATT GGAACTAGCT AGTAGGAAGG GAAG ATG A - #AG TCG       ATG     236                                                                                       - #                  - #             Met Lys Ser -        #Met                                                                                              - #                  - #               1                     - - CTT GTG GTG ACA ATA TCA ATA TGG CTC ATT CT - #T GCA CCA ACT TCA        ACT      284                                                                    Leu Val Val Thr Ile Ser Ile Trp Leu Ile Le - #u Ala Pro Thr Ser Thr            5                - #  10                - #  15                - #  20       - - TGG GCT GTG AAT ACA ATC ATC TAC AAT GTT GG - #A AGT ACC ACC ATT AGC          332                                                                       Trp Ala Val Asn Thr Ile Ile Tyr Asn Val Gl - #y Ser Thr Thr Ile Ser                            25 - #                 30 - #                 35              - - AAA TAC GCC ACT TTT CTG AAT GAT CTT CGT AA - #T GAA GCG AAA GAT CCA          380                                                                       Lys Tyr Ala Thr Phe Leu Asn Asp Leu Arg As - #n Glu Ala Lys Asp Pro                        40     - #             45     - #             50                  - - AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG CC - #C AAT ACA AAT ACA AAT          428                                                                       Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu Pr - #o Asn Thr Asn Thr Asn                    55         - #         60         - #         65                      - - CCA AAG TAC GTG TTG GTT GAG CTC CAA GGT TC - #A AAT AAA AAA ACC ATC          476                                                                       Pro Lys Tyr Val Leu Val Glu Leu Gln Gly Se - #r Asn Lys Lys Thr Ile                70             - #     75             - #     80                          - - ACA CTA ATG CTG AGA CGA AAC AAT TTG TAT GT - #G ATG GGT TAT TCT GAT          524                                                                       Thr Leu Met Leu Arg Arg Asn Asn Leu Tyr Va - #l Met Gly Tyr Ser Asp            85                 - # 90                 - # 95                 - #100       - - CCC TTT GAA ACC AAT AAA TGT CGT TAC CAT AT - #C TTT AAT GAT ATC TCA          572                                                                       Pro Phe Glu Thr Asn Lys Cys Arg Tyr His Il - #e Phe Asn Asp Ile Ser                           105  - #               110  - #               115              - - GGT ACT GAA CGC CAA GAT GTA GAG ACT ACT CT - #T TGC CCA AAT GCC AAT          620                                                                       Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Le - #u Cys Pro Asn Ala Asn                       120      - #           125      - #           130                  - - TCT CGT GTT AGT AAA AAC ATA AAC TTT GAT AG - #T CGA TAT CCA ACA TTG          668                                                                       Ser Arg Val Ser Lys Asn Ile Asn Phe Asp Se - #r Arg Tyr Pro Thr Leu                   135          - #       140          - #       145                      - - GAA TCA AAA GCG GGA GTA AAA TCA AGA AGT CA - #G GTC CAA CTG GGA ATT          716                                                                       Glu Ser Lys Ala Gly Val Lys Ser Arg Ser Gl - #n Val Gln Leu Gly Ile               150              - #   155              - #   160                          - - CAA ATA CTC GAC AGT AAT ATT GGA AAG ATT TC - #T GGA GTG ATG TCA TTC          764                                                                       Gln Ile Leu Asp Ser Asn Ile Gly Lys Ile Se - #r Gly Val Met Ser Phe           165                 1 - #70                 1 - #75                 1 -      #80                                                                              - - ACT GAG AAA ACC GAA GCC GAA TTC CTA TTG GT - #A GCC ATA CAA ATG        GTA      812                                                                    Thr Glu Lys Thr Glu Ala Glu Phe Leu Leu Va - #l Ala Ile Gln Met Val                          185  - #               190  - #               195              - - TCA GAG GCA GCA AGA TTC AAG TAC ATA GAG AA - #T CAG GTG AAA ACT AAT          860                                                                       Ser Glu Ala Ala Arg Phe Lys Tyr Ile Glu As - #n Gln Val Lys Thr Asn                       200      - #           205      - #           210                  - - TTT AAC AGA GCA TTC AAC CCT AAT CCC AAA GT - #A CTT AAT TTG CAA GAG          908                                                                       Phe Asn Arg Ala Phe Asn Pro Asn Pro Lys Va - #l Leu Asn Leu Gln Glu                   215          - #       220          - #       225                      - - ACA TGG GGT AAG ATT TCA ACA GCA ATT CAT GA - #T GCC AAG AAT GGA GTT          956                                                                       Thr Trp Gly Lys Ile Ser Thr Ala Ile His As - #p Ala Lys Asn Gly Val               230              - #   235              - #   240                          - - TTA CCC AAA CCT CTC GAG CTA GTG GAT GCC AG - #T GGT GCC AAG TGG ATA         1004                                                                       Leu Pro Lys Pro Leu Glu Leu Val Asp Ala Se - #r Gly Ala Lys Trp Ile           245                 2 - #50                 2 - #55                 2 -      #60                                                                              - - GTG TTG AGA GTG GAT GAA ATC AAG CCT GAT GT - #A GCA CTC TTA AAC        TAC     1052                                                                    Val Leu Arg Val Asp Glu Ile Lys Pro Asp Va - #l Ala Leu Leu Asn Tyr                          265  - #               270  - #               275              - - GTT GGT GGG AGC TGT CAG ACA ACT TAT AAC CA - #A AAT GCC ATG TTT CCT         1100                                                                       Val Gly Gly Ser Cys Gln Thr Thr Tyr Asn Gl - #n Asn Ala Met Phe Pro                       280      - #           285      - #           290                  - - CAA CTT ATA ATG TCT ACT TAT TAT AAT TAC AT - #G GTT AAT CTT GGT GAT         1148                                                                       Gln Leu Ile Met Ser Thr Tyr Tyr Asn Tyr Me - #t Val Asn Leu Gly Asp                   295          - #       300          - #       305                      - - CTA TTT GAA GGA TTC TGATCATAAA CATAATAAGG AGTATATAT - #A TATTACTCCA         1203                                                                       Leu Phe Glu Gly Phe                                                               310                                                                        - - ACTATATTAT AAAGCTTAAA TAAGAGGCCG TGTTAATTAG TACTTGTTGC CT -             #TTTGCTTT   1263                                                                 - - ATGGTGTTGT TTATTATGCC TTGTATGCTT GTAATATTAT CTAGAGAACA AG -            #ATGTACTG   1323                                                                 - - TGTAATAGTC TTGTTTGAAA TAAAACTTCC AATTATGATG CAAAAAAAAA AA - #AAAA           1379                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 313 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Lys Ser Met Leu Val Val Thr Ile Ser Il - #e Trp Leu Ile Leu Ala        1               5 - #                 10 - #                 15              - - Pro Thr Ser Thr Trp Ala Val Asn Thr Ile Il - #e Tyr Asn Val Gly Ser                   20     - #             25     - #             30                  - - Thr Thr Ile Ser Lys Tyr Ala Thr Phe Leu As - #n Asp Leu Arg Asn Glu               35         - #         40         - #         45                      - - Ala Lys Asp Pro Ser Leu Lys Cys Tyr Gly Il - #e Pro Met Leu Pro Asn           50             - #     55             - #     60                          - - Thr Asn Thr Asn Pro Lys Tyr Val Leu Val Gl - #u Leu Gln Gly Ser Asn       65                 - # 70                 - # 75                 - # 80       - - Lys Lys Thr Ile Thr Leu Met Leu Arg Arg As - #n Asn Leu Tyr Val Met                       85 - #                 90 - #                 95              - - Gly Tyr Ser Asp Pro Phe Glu Thr Asn Lys Cy - #s Arg Tyr His Ile Phe                  100      - #           105      - #           110                  - - Asn Asp Ile Ser Gly Thr Glu Arg Gln Asp Va - #l Glu Thr Thr Leu Cys              115          - #       120          - #       125                      - - Pro Asn Ala Asn Ser Arg Val Ser Lys Asn Il - #e Asn Phe Asp Ser Arg          130              - #   135              - #   140                          - - Tyr Pro Thr Leu Glu Ser Lys Ala Gly Val Ly - #s Ser Arg Ser Gln Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Leu Gly Ile Gln Ile Leu Asp Ser Asn Il - #e Gly Lys Ile Ser        Gly                                                                                             165  - #               170  - #               175             - - Val Met Ser Phe Thr Glu Lys Thr Glu Ala Gl - #u Phe Leu Leu Val Ala                  180      - #           185      - #           190                  - - Ile Gln Met Val Ser Glu Ala Ala Arg Phe Ly - #s Tyr Ile Glu Asn Gln              195          - #       200          - #       205                      - - Val Lys Thr Asn Phe Asn Arg Ala Phe Asn Pr - #o Asn Pro Lys Val Leu          210              - #   215              - #   220                          - - Asn Leu Gln Glu Thr Trp Gly Lys Ile Ser Th - #r Ala Ile His Asp Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys Asn Gly Val Leu Pro Lys Pro Leu Glu Le - #u Val Asp Ala Ser        Gly                                                                                             245  - #               250  - #               255             - - Ala Lys Trp Ile Val Leu Arg Val Asp Glu Il - #e Lys Pro Asp Val Ala                  260      - #           265      - #           270                  - - Leu Leu Asn Tyr Val Gly Gly Ser Cys Gln Th - #r Thr Tyr Asn Gln Asn              275          - #       280          - #       285                      - - Ala Met Phe Pro Gln Leu Ile Met Ser Thr Ty - #r Tyr Asn Tyr Met Val          290              - #   295              - #   300                          - - Asn Leu Gly Asp Leu Phe Glu Gly Phe                                      305                 3 - #10                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1379 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 225..1163                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 225..290                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CTATGAAGTC GGGTCAAAGC ATATACAGGC TATGCATTGT TAGAAACATT GA -             #TGCCTCTG     60                                                                 - - ATCCCGATAA ACAATACAAA TTAGACAATA AGATGACATA CAAGTACCTA AA -            #CTGTGTAT    120                                                                 - - GGGGGAGTGA AACCTCAGCT GCTAAAAAAA CGTTGTAAGA AAAAAAGAAA GT -            #TGTGAGTT    180                                                                 - - AACTACAGGG CGAAAGTATT GGAACTAGCT AGTAGGAAGG GAAG ATG A - #AG TCA       ATG     236                                                                                       - #                  - #             Met Lys Ser -        #Met                                                                                              - #                  - #               1                     - - CTT GTG GTG ACA ATA TCA ATA TGG CTC ATT CT - #T GCA CCA ACT TCA        ACT      284                                                                    Leu Val Val Thr Ile Ser Ile Trp Leu Ile Le - #u Ala Pro Thr Ser Thr            5                - #  10                - #  15                - #  20       - - TGG GCT GTG AAT ACA ATC ATC TAC AAT GTT GG - #A AGT ACC ACC ATT AGC          332                                                                       Trp Ala Val Asn Thr Ile Ile Tyr Asn Val Gl - #y Ser Thr Thr Ile Ser                            25 - #                 30 - #                 35              - - AAA TAC GCC ACT TTT CGG AAT GAT CTT CGT AA - #T GAA GCG AAA GAT CCA          380                                                                       Lys Tyr Ala Thr Phe Arg Asn Asp Leu Arg As - #n Glu Ala Lys Asp Pro                        40     - #             45     - #             50                  - - AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG CC - #C AAT ACA AAT ACA AAT          428                                                                       Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu Pr - #o Asn Thr Asn Thr Asn                    55         - #         60         - #         65                      - - CCA AAG CAC GTG TTG GTT GAG CTC CAA GGT TC - #A AAT AAA AAA ACC ATC          476                                                                       Pro Lys His Val Leu Val Glu Leu Gln Gly Se - #r Asn Lys Lys Thr Ile                70             - #     75             - #     80                          - - ACA CTA ATG CTG AGA CGA AAC AAT TTG TAT GT - #G ATG GGT TAT TCT GAT          524                                                                       Thr Leu Met Leu Arg Arg Asn Asn Leu Tyr Va - #l Met Gly Tyr Ser Asp            85                 - # 90                 - # 95                 - #100       - - CCC TTT GAA ACC AAT AAA TGT CGT TAC CAT AT - #C TTT AAT GAT ATC TCA          572                                                                       Pro Phe Glu Thr Asn Lys Cys Arg Tyr His Il - #e Phe Asn Asp Ile Ser                           105  - #               110  - #               115              - - GGT ACT GAA CGC CAA GAT GTA GAG ACT ACT CT - #T TGC CCA AAT GCC AAT          620                                                                       Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Le - #u Cys Pro Asn Ala Asn                       120      - #           125      - #           130                  - - TCT CGT GTT AGT AAA AAC ATA AAC TTT GAT AG - #T CGA TAT CCA ACA TTG          668                                                                       Ser Arg Val Ser Lys Asn Ile Asn Phe Asp Se - #r Arg Tyr Pro Thr Leu                   135          - #       140          - #       145                      - - GAA TCA AAA GCG GGA GTA AAA TCA AGA AGT CA - #G GTC CAA CTG GGA ATT          716                                                                       Glu Ser Lys Ala Gly Val Lys Ser Arg Ser Gl - #n Val Gln Leu Gly Ile               150              - #   155              - #   160                          - - CAA ATA CTC GAC AGT AAT ATT GGA AAG ATT TC - #T GGA GTG ATG TCA TTC          764                                                                       Gln Ile Leu Asp Ser Asn Ile Gly Lys Ile Se - #r Gly Val Met Ser Phe           165                 1 - #70                 1 - #75                 1 -      #80                                                                              - - ACT GAG AAA ACC GAA GCC GAA TTC CTA TTG GT - #A GCC ATA CAA ATG        GTA      812                                                                    Thr Glu Lys Thr Glu Ala Glu Phe Leu Leu Va - #l Ala Ile Gln Met Val                          185  - #               190  - #               195              - - TCA GAG GCA GCA AGA TTC AAG TAC ATA GAG AA - #T CAG GTG AAA ACT AAT          860                                                                       Ser Glu Ala Ala Arg Phe Lys Tyr Ile Glu As - #n Gln Val Lys Thr Asn                       200      - #           205      - #           210                  - - TTT AAC AGA GCA TTC AAC CCT AAT CCC AAA GT - #A CTT AAT TTG CAA GAG          908                                                                       Phe Asn Arg Ala Phe Asn Pro Asn Pro Lys Va - #l Leu Asn Leu Gln Glu                   215          - #       220          - #       225                      - - ACA TGG GGT AAG ATT TCA ACA GCA ATT CAT GA - #T GCC AAG AAT GGA GTT          956                                                                       Thr Trp Gly Lys Ile Ser Thr Ala Ile His As - #p Ala Lys Asn Gly Val               230              - #   235              - #   240                          - - TTA CCC AAA CCT CTC GAG CTA GTG GAT GCC AG - #T GGT GCC AAG TGG ATA         1004                                                                       Leu Pro Lys Pro Leu Glu Leu Val Asp Ala Se - #r Gly Ala Lys Trp Ile           245                 2 - #50                 2 - #55                 2 -      #60                                                                              - - GTG TTG AGA GTG GAT GAA ATC AAG CCT GAT GT - #A GCA CTC TTA AAC        TAC     1052                                                                    Val Leu Arg Val Asp Glu Ile Lys Pro Asp Va - #l Ala Leu Leu Asn Tyr                          265  - #               270  - #               275              - - GTT GGT GGG AGC TGT CAG ACA ACT TAT AAC CA - #A AAT GCC ATG TTT CCT         1100                                                                       Val Gly Gly Ser Cys Gln Thr Thr Tyr Asn Gl - #n Asn Ala Met Phe Pro                       280      - #           285      - #           290                  - - CAA CTT ATA ATG TCT ACT TAT TAT AAT TAC AT - #G GTT AAT CTT GGT GAT         1148                                                                       Gln Leu Ile Met Ser Thr Tyr Tyr Asn Tyr Me - #t Val Asn Leu Gly Asp                   295          - #       300          - #       305                      - - CTA TTT GAA GGA TTC TGATCATAAA CATAATAAGG AGTATATAT - #A TATTACTCCA         1203                                                                       Leu Phe Glu Gly Phe                                                               310                                                                        - - ACTATATTAT AAAGCTTAAA TAAGAGGCCG TGTTAATTAG TACTTGTTGC CT -             #TTTGCTTT   1263                                                                 - - ATGGTGTTGT TTATTATGCC TTGTATGCTT GTAATATTAT CTAGAGAACA AG -            #ATGTACTG   1323                                                                 - - TGTAATAGTC TTGTTTGAAA TAAAACTTCC AATTATGATG CAAAAAAAAA AA - #AAAA           1379                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 313 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Lys Ser Met Leu Val Val Thr Ile Ser Il - #e Trp Leu Ile Leu Ala        1               5 - #                 10 - #                 15              - - Pro Thr Ser Thr Trp Ala Val Asn Thr Ile Il - #e Tyr Asn Val Gly Ser                   20     - #             25     - #             30                  - - Thr Thr Ile Ser Lys Tyr Ala Thr Phe Arg As - #n Asp Leu Arg Asn Glu               35         - #         40         - #         45                      - - Ala Lys Asp Pro Ser Leu Lys Cys Tyr Gly Il - #e Pro Met Leu Pro Asn           50             - #     55             - #     60                          - - Thr Asn Thr Asn Pro Lys His Val Leu Val Gl - #u Leu Gln Gly Ser Asn       65                 - # 70                 - # 75                 - # 80       - - Lys Lys Thr Ile Thr Leu Met Leu Arg Arg As - #n Asn Leu Tyr Val Met                       85 - #                 90 - #                 95              - - Gly Tyr Ser Asp Pro Phe Glu Thr Asn Lys Cy - #s Arg Tyr His Ile Phe                  100      - #           105      - #           110                  - - Asn Asp Ile Ser Gly Thr Glu Arg Gln Asp Va - #l Glu Thr Thr Leu Cys              115          - #       120          - #       125                      - - Pro Asn Ala Asn Ser Arg Val Ser Lys Asn Il - #e Asn Phe Asp Ser Arg          130              - #   135              - #   140                          - - Tyr Pro Thr Leu Glu Ser Lys Ala Gly Val Ly - #s Ser Arg Ser Gln Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Leu Gly Ile Gln Ile Leu Asp Ser Asn Il - #e Gly Lys Ile Ser        Gly                                                                                             165  - #               170  - #               175             - - Val Met Ser Phe Thr Glu Lys Thr Glu Ala Gl - #u Phe Leu Leu Val Ala                  180      - #           185      - #           190                  - - Ile Gln Met Val Ser Glu Ala Ala Arg Phe Ly - #s Tyr Ile Glu Asn Gln              195          - #       200          - #       205                      - - Val Lys Thr Asn Phe Asn Arg Ala Phe Asn Pr - #o Asn Pro Lys Val Leu          210              - #   215              - #   220                          - - Asn Leu Gln Glu Thr Trp Gly Lys Ile Ser Th - #r Ala Ile His Asp Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys Asn Gly Val Leu Pro Lys Pro Leu Glu Le - #u Val Asp Ala Ser        Gly                                                                                             245  - #               250  - #               255             - - Ala Lys Trp Ile Val Leu Arg Val Asp Glu Il - #e Lys Pro Asp Val Ala                  260      - #           265      - #           270                  - - Leu Leu Asn Tyr Val Gly Gly Ser Cys Gln Th - #r Thr Tyr Asn Gln Asn              275          - #       280          - #       285                      - - Ala Met Phe Pro Gln Leu Ile Met Ser Thr Ty - #r Tyr Asn Tyr Met Val          290              - #   295              - #   300                          - - Asn Leu Gly Asp Leu Phe Glu Gly Phe                                      305                 3 - #10                                                  __________________________________________________________________________

What is claimed is:
 1. A DNA molecule comprising a sequence encoding apokeweed antiviral protein (PAP) mutant having reduced phytotoxicitycompared to mature, wild-type PAP or PAP-v (Leu20Arg, Tyr49His), saidPAP mutant contain intact catalytic active site amino acid residues(Glu176, Arg179) but differing from wild-type PAP substantially in thatit is truncated at its N-terminus from 1 to about 38 amino acidresidues, wherein the PAP mutant encoded by said DNA exhibits anti-viralor anti-fungal activity in plants.
 2. The DNA molecule of claim 1wherein said sequences encodes a PAP mutant selected from the group ofPAP mutants consisting of PAP (2-262), PAP (3-262), PAP (4-262), PAP(5-262), PAP (6-262), PA) (7-262), PAP (8-262), PAP (9-262), PAP(10-262), PAP (11-262), PAP (12-262), PAP (13-212), PAP (14-262), PAP(15-262), PAP (16-262), PAP (17-262), PAP (18-262), PAP (19-262), PAP(20-262), PAP (21-262), PAP (22-262), PAP (23-262), PAP (24-262), PAP(25-262), PAP 26-262), PAP (27-262), PAP (28-262), PAP (29-262), PAP(30-262), PAP (31-262), PAP (32-2), PAP (33-262), PAP (34-262), PAP(35-262), PAP (36-262), PAP (37-262), PAP (38-262) an PAP (39-262). 3.The DNA molecule of claim 1 wherein said PAP mutant comprises theN-terminal signal sequence of wild-type PAP.
 4. The DNA molecule ofclaim 1 wherein said PAP mutant comprises the C-terminal extension ofwild-type PAP.
 5. The DNA molecule of claim 1 further comprising apromoter operably linked to said sequence.
 6. The DNA molecule of claim5 wherein said promoter is functional in a yeast cell.
 7. Therecombinant DNA molecule of claim 5 wherein said promoter is functionalin a plant cell.
 8. The recombinant DNA molecule of claim 5 wherein saidpromoter is an inducible promoter.
 9. The recombinant DNA molecule ofclaim 5 wherein said promoter is constitutive promoter.
 10. Arecombinant vector comprising the DNA molecule of claim
 1. 11. Arecombinant protoplast stably transformed with the DNA molecule orclaim
 1. 12. A host cell stably transformed with the DNA molecule ofclaim
 1. 13. The host cell of claim 12 which is an E. coli cell.
 14. Thehost cell of claim 12 which is a yeast cell.
 15. The yeast cell of claim14 which is a Saccharomyces cerevisiae cell.
 16. The host cell of claim12 which is a plant cell.
 17. The host cell of claim 12 wherein said DNAmolecule is operably linked to an inducible promoter functional in saidhost cell.
 18. A transgenic plant regenerated from the protoplast ofclaim
 11. 19. A transgenic plant comprising the DNA molecule of claim 1,wherein the sequence is expressed.
 20. The transgenic plant of claim 18,which is a monocot plant.
 21. The transgenic plant of claim 19, whereinsaid monocot is a cereal crop plant.
 22. The transgenic plant of claim18, which is a dicot plant.
 23. Seed derived from the transgenic plantof claim 18 wherein the genome of said seed comprises the DNA ofclaim
 1. 24. A method of making a plant that has increased resistance toviruses and/or fungi, comprising preparing a plant having a genome thatcontains the DNA molecule of claim 1 wherein said sequence is expressed.25. The method of claim 23 comprising stably transforming a protoplastwith the DNA molecule, and generating the plant from the transformedprotoplast.
 26. The method of claim 23 comprising introducing the DNAmolecule into plant tissue, and regenerating the plant from the planttissue containing the DNA molecule.